MAGE-A3 peptides presented by HLA class II molecules

ABSTRACT

The invention describes HLA class II binding peptides encoded by the MAGE-A3 tumor associated gene, as well as nucleic acids encoding such peptides and antibodies relating thereto. The peptides stimulate the activity and proliferation of CD4 +  T lymphocytes. Methods and products also are provided for diagnosing and treating conditions characterized by expression of the MAGE-A3 gene.

FIELD OF THE INVENTION

[0001] This invention relates to fragments of the tumor associated geneproduct MAGE-A3 which bind to and are presented to T lymphocytes by HLAclass II molecules. The peptides, nucleic acid molecules which code forsuch peptides, as well as related antibodies and CD4⁺ T lymphocytes, areuseful, inter alia, in diagnostic and therapeutic contexts.

BACKGROUND OF THE INVENTION

[0002] The process by which the mammalian immune system recognizes andreacts to foreign or alien materials is complex. An important facet ofthe system is the T cell response, which in part comprises mature Tlymphocytes which are positive for either CD4 or CD8 cell surfaceproteins. T cells can recognize and interact with other cells via cellsurface complexes on the other cells of peptides and molecules referredto as human leukocyte antigens (“HLAs”) or major histocompatibilitycomplexes (“MHCs”). The peptides are derived from larger molecules whichare processed by the cells which also present the HLA/MHC molecule. SeeMale et al., Advanced Immunology (J. P. Lipincott Company, 1987),especially chapters 6-10. The interaction of T cells and complexes ofHLA/peptide is restricted, requiring a specific T cell for a specificcomplex of an HLA molecule and a peptide. If a specific T cell is notpresent, there is no T cell response even if its partner complex ispresent. Similarly, there is no response if the specific complex isabsent, but the T cell is present. The mechanisms described above areinvolved in the immune system's response to foreign materials, inautoimmune pathologies, and in responses to cellular abnormalities.

[0003] The T cell response to foreign antigens includes both cytolytic Tlymphocytes and helper T lymphocytes. CD8⁺ cytotoxic or cytolytic Tcells (CTLs) are T cells which, when activated, lyse cells that presentthe appropriate antigen presented by HLA class I molecules. CD4⁺ Thelper cells are T cells which secrete cytokines to stimulatemacrophages and antigen-producing B cells which present the appropriateantigen by HLA class II molecules on their surface.

[0004] The mechanism by which T cells recognize alien materials also hasbeen implicated in cancer. A number of cytolytic T lymphocyte (CTL)clones directed against autologous melanoma have been described. In someinstances, the antigens recognized by these clones have beencharacterized. In De Plaen et al., Immunogenetics 40:360-369 (1994), the“MAGE” family, a family of genes encoding tumor specific antigens, isdescribed. (See also PCT application PCT/US92/04354, published on Nov.26, 1992.) The expression products of these genes are processed intopeptides which, in turn, are expressed on cell surfaces. This can leadto lysis of the tumor cells by specific CTLs. The genes are said to codefor “tumor rejection antigen precursors” or “TRAP” molecules, and thepeptides derived therefrom are referred to as “tumor rejection antigens”or “TRAs”. See Traversari et al., Immunogenetics 35: 145 (1992); van derBruggen et al., Science 254: 1643 (1991), for further information onthis family of genes. Also, see U.S. Pat. No. 5,342,774.

[0005] In U.S. Pat. No. 5,405,940, MAGE nonapeptides are taught whichare presented by the HLA-A1 molecule. Given the known specificity ofparticular peptides for particular HLA molecules, one should expect aparticular peptide to bind one HLA molecule, but not others. This isimportant, because different individuals possess different HLAphenotypes. As a result, while identification of a particular peptide asbeing a partner for a specific HLA molecule has diagnostic andtherapeutic ramifications, these are only relevant for individuals withthat particular HLA phenotype. There is a need for further work in thearea, because cellular abnormalities are not restricted to oneparticular HLA phenotype, and targeted therapy requires some knowledgeof the phenotype of the abnormal cells at issue.

[0006] In U.S. Pat. No. 5,591,430, additional isolated MAGE-A3 peptidesare taught which are presented by the HLA-A2 molecule. Therefore, agiven TRAP can yield a plurality of TRAs.

[0007] The foregoing references describe isolation and/orcharacterization of tumor rejection antigens which are presented by HLAclass I molecules. These TRAs can induce activation and proliferation ofCD8⁺ cytotoxic T lymphocytes (CTLs) which recognize tumor cells thatexpress the tumor associated genes (e.g. MAGE genes) which encode theTRAs.

[0008] The importance of CD4⁺ T lymphocytes (helper T cells) inantitumor immunity has been demonstrated in animal models in which thesecells not only serve cooperative and effector functions, but are alsocritical in maintaining immune memory (reviewed by Topalian, Curr. Opin.Immunol. 6:741-745, 1994). Moreover, several studies support thecontention that poor tumor-specific immunity is due to inadequateactivation of T helper cells.

[0009] It has recently been demonstrated that the tyrosinase geneencodes peptides which are presented by HLA class II molecules tostimulate CD4⁺ T lymphocytes (Topalian et al., 1994; Yee et al., J.Immunol. 157:4079-4086, 1996; Topalian et al., J. Exp. Med.183:1965-1971, 1996). As with many cancer associated antigens,tyrosinase is expressed in a limited percentage of tumors and in limitedtypes of tumors. Furthermore, the two identified MHC class II bindingtyrosinase peptides are HLA-DRB1*0401-restricted peptides, recognizedonly by cells which express the particular HLA molecule.

[0010] More recently, HLA class II peptide have been identified in theMAGE-A3, a cancer-testis antigen widely expressed in cancer cells butnot in normal cells except testis. See U.S. Pat. No. 5,965,535 andPCT/US99/21230.

[0011] Although the cancer antigens tyrosinase and MAGE-A3 have beenshown to contain HLA class II binding peptides, there exist manypatients who would not benefit from any therapy which includes helper Tcell stimulation via the aforementioned tyrosinase and MAGE-A3 peptides,either because the patient's tumor does not express tyrosinase, orbecause the patient does not express the appropriate HLA molecule.Accordingly, there is a need for the identification of additional tumorassociated antigens which contain epitopes presented by MHC class IImolecules and recognized by CD4⁺ lymphocytes.

SUMMARY OF THE INVENTION

[0012] It now has been discovered that the MAGE-A3 gene encodesadditional HLA class II binding peptides that are epitopes presented byHLA-DR1. These peptides, when presented by an antigen presenting cellhaving the appropriate HLA class II molecule, effectively induce theactivation and proliferation of CD4⁺ T lymphocytes.

[0013] The invention provides isolated MAGE-A3 peptides which bind HLAclass II molecules, and functional variants of such peptides, thefunctional variants comprising one or more amino acid additions,substitutions or deletions to the MAGE-A3 peptide sequence. Theinvention also provides isolated nucleic acid molecules encoding suchpeptides, expression vectors containing those nucleic acid molecules,host cells transfected with those nucleic acid molecules, and antibodiesto those peptides and complexes of the peptides and HLA class II antigenpresenting molecules. T lymphocytes which recognize complexes of thepeptides and HLA class II antigen presenting molecules are alsoprovided. Kits and vaccine compositions containing the foregoingmolecules additionally are provided. The foregoing can be used in thediagnosis or treatment of conditions characterized by the expression ofMAGE-A3. As it is known that the members of the MAGE family ofpolypeptides and nucleic acids share significant sequence identity andfunctional homology (e.g., as tumor antigens and precursors), theinvention also embraces HLA binding peptides of similar amino acidsequence derived from members of the MAGE family other than MAGE-A3.Therefore, it is understood that the disclosure contained herein ofMAGE-A3 HLA class II binding peptides, compositions containing suchpeptides, and methods of identifying and using such peptides appliesalso to other members of the MAGE tumor associated antigen family.

[0014] According to one aspect of the invention, isolated MAGE-A3 HLAclass II-binding peptides are provided. The peptides include an aminoacid sequence selected from the group consisting of SEQ ID NO:3, SEQ IDNO:4 and SEQ ID NO:7, or a functional variant thereof comprising 1-10amino acid additions, substitutions or deletions. However, the isolatedMAGE-A3 HLA class II binding peptide does not include the full lengthMAGE-A3 protein sequence, e.g., the amino acid sequence of SEQ ID NO:2.

[0015] In certain embodiments, the isolated HLA class II-bindingpeptides consist essentially of an amino acid sequence selected from thegroup consisting of SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:7, or consistof an amino acid sequence selected from the group consisting of SEQ IDNO:3, SEQ ID NO:4, SEQ ID NO:7 and SEQ ID NO:16. In preferredembodiments, the isolated HLA class II-binding peptide consists of theamino acid sequence set forth as SEQ ID NO:4.

[0016] In other embodiments, the isolated MAGE-A3 HLA class II-bindingpeptides include an endosomal targeting signal, which preferably is anendosomal targeting portion of human invariant chain Ii. In still otherembodiments, the isolated MAGE-A3 HLA class II-binding peptides arenon-hydrolyzable. Preferred non-hydrolyzable peptides include peptidescomprising D-amino acids, peptides comprising a -psi[CH₂NH]-reducedamide peptide bond, peptides comprising a -psi[COCH₂]-ketomethylenepeptide bond, peptides comprising a -psi[CH(CN)NH]-(cyanomethylene)aminopeptide bond, peptides comprising a -psi [CH₂CH(OH)]-hydroxyethylenepeptide bond, peptides comprising a -psi[CH₂O]-peptide bond, andpeptides comprising a -psi[CH₂S]-thiomethylene peptide bond.

[0017] According to another aspect of the invention, compositionscomprising one or more isolated HLA class I-binding peptides and one ormore isolated MAGE-A3 HLA class II-binding peptides are provided. Inthese compositions, at least one of the isolated MAGE-A3 HLA classII-binding peptides includes an amino acid sequence selected from thegroup consisting of SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:7, or afunctional variant thereof. The HLA class II binding peptide does notinclude a full length MAGE-A3 protein.

[0018] In some embodiments, the HLA class I-binding peptides and theMAGE-A3 HLA class II-binding peptides are combined as a polytopepolypeptide, e.g., a series of contiguous epitope amino acid sequences,optionally connected by linker (non-epitope) amino acid sequences.Preferably the isolated MAGE-A3 HLA class II-binding peptides consistessentially of an amino acid sequence selected from the group consistingof SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:7. In other preferredembodiments, the isolated MAGE-A3 HLA class II-binding peptided consistof an amino acid sequence selected from the group consisting of SEQ IDNO:3, SEQ ID NO:4, SEQ ID NO:7 and SEQ ID NO:16. Most preferably, theisolated MAGE-A3 HLA class II-binding peptide consists of the amino acidsequence set forth as SEQ ID NO:4.

[0019] In certain embodiments, the isolated MAGE-A3 HLA class II-bindingpeptides include an endosomal targeting signal, which preferablyincludes an endosomal targeting portion of human invariant chain Ii.

[0020] According to still another aspect of the invention, compositionsare provided, which include one or more of the foregoing isolatedMAGE-A3 HLA class II-binding peptides complexed with one or moreisolated HLA class II molecules. In certain embodiments, the number ofisolated MAGE-A3 HLA class II-binding peptides and the number ofisolated HLA class II molecules are equal. Preferably the isolatedMAGE-A3 HLA class II-binding peptides and the isolated MAGE-A3 HLA classII molecules are coupled as a tetrameric molecule of individual isolatedMAGE-A3 HLA class II-binding peptides bound to individual isolated HLAclass II molecules. In the foregoing embodiments, the HLA class IImolecules preferably are DR1 molecules.

[0021] According to a further aspect of the invention, isolated nucleicacids are provided that encode one or more of the foregoing MAGE-A3 HLAclass II-binding peptides. The nucleic acid molecules do not encode fulllength MAGE-A3 protein. In preferred embodiments the nucleic acidscomprise a nucleotide sequence selected from the group consisting of SEQID NO:5, SEQ ID NO:6 and SEQ ID NO:15.

[0022] Expression vectors that include the foregoing isolated nucleicacids operably linked to a promoter also are provided in accordance withthe invention. In the expression vectors, the nucleic acid preferablyincludes a nucleotide sequence set forth as SEQ ID NO:5, SEQ ID NO:6 orSEQ ID NO:15. Optionally the expression vectors include a nucleic acidwhich encodes an HLA-DR1 molecule.

[0023] According to a further aspect of the invention, host cells areprovided that are transfected or transformed with one or more of theforegoing expression vectors. Some of the host cells express HLA-DR1molecules.

[0024] According to yet another aspect of the invention, methods forenriching selectively a population of T lymphocytes with CD4⁺ Tlymphocytes specific for a MAGE-A3 HLA class II-binding peptide areprovided. The methods include contacting an isolated population of Tlymphocytes with an agent presenting a complex of the MAGE-A3 HLA classII-binding peptide and an HLA class II molecule in an amount sufficientto selectively enrich the isolated population of T lymphocytes with theCD4⁺ T lymphocytes. The MAGE-A3 HLA class II-binding peptide comprisesan amino acid sequence selected from the group consisting of SEQ IDNO:3, SEQ ID NO:4 and SEQ ID NO:7, or a functional variant thereof.

[0025] In certain embodiments, the MAGE-A3 HLA class II-binding peptidesconsist essentially of an amino acid sequence selected from the groupconsisting of SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:7. In otherembodiments, the MAGE-A3 HLA class II-binding peptides consist of anamino acid sequence selected from the group consisting of SEQ ID NO:3,SEQ ID NO: 4, SEQ ID NO:7 and SEQ ID NO:16. Preferably, the MAGE-A3 HLAclass II-binding peptide consists of the amino acid sequence set forthas SEQ ID NO:4. In still other embodiments, the HLA class II molecule isan HLA-DR1 molecule. The MAGE-A3 HLA class II binding peptide caninclude an endosomal targeting portion of human invariant chain Ii.

[0026] According to another aspect of the invention, methods fordiagnosing a disorder characterized by expression of MAGE-A3 areprovided. The methods include contacting a biological sample isolatedfrom a subject with an agent that is specific for the MAGE-A3 HLA classII binding peptide, and determining the interaction between the agentand the MAGE-A3 HLA class II binding peptide as a determination of thedisorder. In these methods, the MAGE-A3 HLA class II-binding peptidecomprises an amino acid sequence selected from the group consisting ofSEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:7, or a functional variantthereof. In preferred methods, the MAGE-A3 HLA class II-binding peptideconsists essentially of an amino acid sequence selected from the groupconsisting of SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:7, or consists ofan amino acid sequence selected from the group consisting of SEQ IDNO:3, SEQ ID NO: 4, SEQ ID NO:7 and SEQ ID NO: 16. Preferably, theMAGE-A3 HLA class II-binding peptide consists of the amino acid sequenceset forth as SEQ ID NO:4. In the foregoing methods, the agent preferablyis an antibody or an antigen binding fragment thereof.

[0027] An additional aspect of the invention provides methods fordiagnosing a disorder characterized by expression of a MAGE-A3 HLA classII-binding peptide, which forms a complex with an HLA class II molecule.The methods include contacting a biological sample isolated from asubject with an agent that binds the complex and determining bindingbetween the complex and the agent as a determination of the disorder. Insuch methods the HLA class II molecule is an HLA-DR1 and the MAGE-A3 HLAclass II-binding peptide comprises an amino acid sequence selected fromthe group consisting of SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:7, or afunctional variant thereof.

[0028] In some embodiments, the MAGE-A3 HLA class II-binding peptideconsists essentially of an amino acid sequence selected from the groupconsisting of SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:7, or consists ofan amino acid sequence selected from the group consisting of SEQ IDNO:3, SEQ ID NO: 4, SEQ ID NO:7 and SEQ ID NO:16. In preferredembodiments, the MAGE-A3 HLA class II-binding peptide consists of theamino acid sequence set forth as SEQ ID NO:4.

[0029] A further aspect of the invention provides methods for treating asubject having a disorder characterized by expression of MAGE-A3. Themethods include administering to the subject an amount of a MAGE-A3 HLAclass II-binding peptide effective to ameliorate the disorder, whereinthe MAGE-A3 HLA class II-binding peptide comprises an amino acidsequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:4and SEQ ID NO:7, or a functional variant thereof.

[0030] In some embodiments, the MAGE-A3 HLA class II binding peptidecomprises an endosomal targeting signal, preferably an endosomaltargeting portion of human invariant chain Ii. In other embodiments, theMAGE-A3 HLA class II-binding peptide consists essentially of an aminoacid sequence selected from the group consisting of SEQ ID NO:3, SEQ IDNO:4 and SEQ ID NO:7, or consists of an amino acid sequence selectedfrom the group consisting of SEQ ID NO:3, SEQ ID NO: 4, SEQ ID NO:7 andSEQ ID NO:16. Preferably, the MAGE-A3 HLA class II-binding peptideconsists of the amino acid sequence set forth as SEQ ID NO:4.

[0031] According to another aspect of the invention, methods fortreating a subject having a disorder characterized by expression ofMAGE-A3 are provided. The methods include administering to the subjectan amount of a HLA class I-binding peptide and an amount of a MAGE-A3HLA class II-binding peptide effective to ameliorate the disorder. Inthese methods, the MAGE-A3 HLA class II-binding peptide comprises anamino acid sequence selected from the group consisting of SEQ ID NO:3,SEQ ID NO:4 and SEQ ID NO:7, or a functional variant thereof.

[0032] In some embodiments the HLA class I-binding peptide and theMAGE-A3 HLA class II-binding peptide are combined as a polytopepolypeptide. In other embodiments, the HLA class I-binding peptide is aMAGE-A3 HLA class I-binding peptide. In still other embodiments, theMAGE-A3 HLA class II binding peptide comprises an endosomal targetingsignal, preferably an endosomal targeting portion of human invariantchain Ii.

[0033] The MAGE-A3 HLA class II-binding peptide, in certain embodimentsconsists essentially of an amino acid sequence selected from the groupconsisting of SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:7, or consists ofan amino acid sequence selected from the group consisting of SEQ IDNO:3, SEQ ID NO:4, SEQ ID NO:7 and SEQ ID NO:16. Preferably the MAGE-A3HLA class II-binding peptide consists of the amino acid sequence setforth as SEQ ID NO:4.

[0034] According to still another aspect of the invention, additionalmethods for treating a subject having a disorder characterized byexpression of MAGE-A3 are provided. The methods include administering tothe subject an amount of an agent which enriches selectively in thesubject the presence of complexes of an HLA class II molecule and aMAGE-A3 HLA class II-binding peptide, sufficient to ameliorate thedisorder. In these methods, the HLA class II molecule is an HLA-DR1molecule and the MAGE-A3 HLA class II-binding peptide comprises an aminoacid sequence selected from the group consisting of SEQ ID NO:3, SEQ IDNO:4 and SEQ ID NO:7, or a functional variant thereof.

[0035] In certain embodiments, the MAGE-A3 HLA class II-binding peptideconsists essentially of an amino acid sequence selected from the groupconsisting of SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:7, or consists ofan amino acid sequence selected from the group consisting of SEQ IDNO:3, SEQ ID NO: 4, SEQ ID NO:7 and SEQ ID NO:16. Preferably, theMAGE-A3 HLA class II-binding peptide consists of the amino acid sequenceset forth as SEQ ID NO:4.

[0036] In other embodiments, the agent comprises a MAGE-A3 HLA class IIbinding peptide. Preferably the MAGE-A3 HLA class II binding peptidecomprises an endosomal targeting signal. More preferably the endosomaltargeting signal comprises an endosomal targeting portion of humaninvariant chain Ii.

[0037] In another aspect of the invention, other methods for treating asubject having a disorder characterized by expression of MAGE-A3 areprovided. The methods include administering to the subject an amount ofautologous CD4⁺ T lymphocytes sufficient to ameliorate the disorder,wherein the CD4⁺ T lymphocytes are specific for complexes of an HLAclass II molecule and a MAGE-A3 HLA class II-binding peptide. In suchmethods the HLA class II molecule is an HLA-DR1 molecule and the MAGE-A3HLA class II-binding peptide comprises an amino acid sequence selectedfrom the group consisting of SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:7,or a functional variant thereof.

[0038] In certain embodiments of these methods the MAGE-A3 HLA classII-binding peptide consists essentially of an amino acid sequenceselected from the group consisting of SEQ ID NO:3, SEQ ID NO:4 and SEQID NO:7, or consists of an amino acid sequence selected from the groupconsisting of SEQ ID NO:3, SEQ ID NO: 4, SEQ ID NO:7 and SEQ ID NO:16.Preferably the MAGE-A3 HLA class II-binding peptide consists of theamino acid sequence set forth as SEQ ID NO:4.

[0039] Methods for identifying functional variants of a MAGE-A3 HLAclass II binding peptide are also provided in another aspect of theinvention. The methods include selecting a MAGE-A3 HLA class II bindingpeptide which comprisies an amino acid sequence selected from the groupconsisting of SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:7, an HLA class IIbinding molecule which binds the MAGE-A3 HLA class II binding peptide,and a T cell which is stimulated by the MAGE-A3 HLA class II bindingpeptide presented by the HLA class II binding molecule. The methods alsoinclude mutating a first amino acid residue of the MAGE-A3 HLA class IIbinding peptide to prepare a variant peptide and determining the bindingof the variant peptide to HLA class II binding molecule and thestimulation of the T cell. Binding of the variant peptide to the HLAclass II binding molecule and stimulation of the T cell by the variantpeptide presented by the HLA class II binding molecule indicates thatthe variant peptide is a functional variant.

[0040] In some embodiments, the foregoing methods also include a step ofcomparing the stimulation of the T cell by the MAGE-A3 HLA class IIbinding peptide and the stimulation of the T cell by the functionalvariant as a determination of the effectiveness of the stimulation ofthe T cell by the functional variant.

[0041] In another aspect of the invention, isolated polypeptides areprovided which binds selectively an isolated MAGE-A3 HLA classII-binding peptide that consists essentially of an amino acid sequenceselected from the group consisting of SEQ ID NO:3, SEQ ID NO:4 and SEQID NO:7, provided that the isolated polypeptide is not an HLA class IImolecule.

[0042] In certain embodiments, the isolated polypeptide is an antibody,preferably a monoclonal antibody. Preferred monoclonal antibodiesinclude human antibodies, humanized antibodies, chimeric antibodies andsingle chain antibodies. In other embodiments, the isolated polypeptideis an antibody fragment. Preferred antibody fragments include Fabfragments, F(ab)₂ fragments, Fv fragments and fragments including a CDR3region selective for a MAGE-A3 HLA class II-binding peptide.

[0043] In another aspect of the invention, isolated CD4⁺ T lymphocyteswhich selectively bind a complex of an HLA class II molecule and aMAGE-A3 HLA class II-binding peptide are provided. The HLA class IImolecule is an HLA-DR1 molecule and wherein the MAGE-A3 HLA classII-binding peptide comprises an amino acid sequence selected from thegroup consisting of SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:7, or afunctional variant thereof. In certain embodiments, the MAGE-A3 HLAclass TI-binding peptide consists essentially of an amino acid sequenceselected from the group consisting of SEQ ID NO:3, SEQ ID NO:4 and SEQID NO:7, or consists of an amino acid sequence selected from the groupconsisting of SEQ ID NO:3, SEQ ID NO: 4, SEQ ID NO:7 and SEQ ID NO:16.Preferably the MAGE-A3 HLA class II-binding peptide consists of theamino acid sequence set forth as SEQ ID NO:4.

[0044] According to yet another aspect of the invention, isolatedantigen presenting cells are provided which include a complex of an HLAclass II molecule and a MAGE-A3 HLA class II-binding peptide. In thesecells the HLA class II molecule is an HLA-DR1 molecule and the MAGE-A3HLA class II-binding peptide includes an amino acid sequence selectedfrom the group consisting of SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:7.In some embodiments, the MAGE-A3 HLA class II-binding peptide consistsessentially of an amino acid sequence selected from the group consistingof SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:7 or consists of an amino acidsequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:7 and SEQ ID NO: 16. Preferably the MAGE-A3 HLA classII-binding peptide consists of the amino acid sequence set forth as SEQID NO:4.

[0045] The invention also provides pharmaceutical preparationscontaining any one or more of the medicaments described above orthroughout the specification. Such pharmaceutical preparations caninclude pharmaceutically acceptable diluent carriers or excipients.

[0046] The use of the foregoing compositions, peptides and nucleic acidsin the preparation of a medicament, particularly a medicament fortreatment of cancer, also is provided.

[0047] These and other objects of the invention will be described infurther detail in connection with the detailed description of theinvention.

BRIEF DESCRIPTION OF THE DRAWINGS

[0048]FIG. 1 shows that a CD4⁺ T cell clone directed against a MAGE-A3derived antigen.

[0049]FIG. 2 is a graph showing titration of the MAGE-A3 peptiderecognized by CD4+clone MAGJ569/F4.3.

[0050]FIG. 3 is a graph showing that the MAGE-A3 peptide is presented toCD4⁺ clone MAGJ569/F4.3 by HLA-DR molecules

[0051]FIG. 4 is a graph depicting recognition of DR1 tumor cellsexpressing MAGE-A3 by CD4⁺ clone MAGJ569/F4.3.

DETAILED DESCRIPTION OF THE INVENTION

[0052] The invention provides isolated MAGE-A3 peptides presented by HLAclass II molecules, which peptides stimulate the proliferation andactivation of CD4⁺ T lymphocytes. Such peptides are referred to hereinas “MAGE-A3 HLA class II binding peptides,” “HLA class II bindingpeptides” and “MHC class II binding peptides.” Hence, one aspect of theinvention is an isolated peptide which includes the amino acid sequenceof SEQ ID NO:4. The peptides referred to herein as “MAGE-A3 HLA class IIbinding peptides” include fragments of MAGE-A3 protein, but do notinclude full-length MAGE-A3 protein. Likewise, nucleic acids that encodethe “MAGE-A3 HLA class II binding peptides” include fragments of theMAGE-A3 gene coding region, but do not include the full-length MAGE-A3coding region.

[0053] The examples below show the isolation of peptides which areMAGE-A3 HLA class II binding peptides. These exemplary peptides areprocessed translation products of the MAGE-A3 nucleic acid (SEQ ID NO:1,the polypeptide sequence of which is given as SEQ ID NO:2). As such, itwill be appreciated by one of ordinary skill in the art that thetranslation products from which a MAGE-A3 HLA class II binding peptideis processed to a final form for presentation may be of any length orsequence so long as they encompass the MAGE-A3 HLA class II bindingpeptide. As demonstrated in the examples below, peptides or proteins assmall as 13 or 14 amino acids and as large as the amino acid sequence ofthe MAGE-A3 protein (SEQ ID NO:2) are appropriately processed, presentedby HLA class II molecules and effective in stimulating CD4⁺ Tlymphocytes. MAGE-A3 HLA class II binding peptides, such as the peptidesof SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:7 may have one, two, three,four, five, six, seven, eight, nine, ten, 15, 20, 25, 30, 40, 50 or moreamino acids added to either or both ends. The antigenic portion of sucha peptide is cleaved out under physiological conditions for presentationby HLA class II molecules. It is also well known in the art that HLAclass II peptide length is variable between about 10 amino acids andabout 30 amino acids (Engelhard, Ann. Rev. Immunol. 12:181-201, 1994).Most of the HLA class II binding peptides fall in to the length range of12-19 amino acids. Nested sets of HLA class II binding peptides havebeen identified, wherein the peptides share a core sequence but havedifferent amino acids at amino and/or carboxyl terminal ends (see, e.g.,Chicz et al., J. Exp. Med. 178:27-47, 1993). Thus additional MAGE-A3 HLAclass II binding peptides, as well as MAGE family HLA class II bindingpeptides, can be identified by one of ordinary skill in the artaccording to the procedures described herein.

[0054] The procedures described in the Examples can be utilized toidentify MAGE family HLA class II binding peptides. Thus, for example,one can load antigen presenting cells, such as dendritic cells of normalblood donors, with a recombinant MAGE protein (or a fragment thereof) bycontacting the cells with the MAGE polypeptide or by introducing intothe cells a nucleic acid molecule which directs the expression of theMAGE protein of interest. The antigen-presenting cells then can be usedto induce in vitro the activation and proliferation of specific CD4lymphocytes which recognize MAGE HLA class II binding peptides. Thesequence of the peptides then can be determined as described in theExamples, e.g., by stimulating cells with peptide fragments of the MAGEprotein used to stimulate the activation and proliferation of CD4lymphocytes. Alternatively, one can load antigen presenting cells withpeptides derived from a MAGE protein. For example, one can makepredictions of peptide sequences derived from MAGE family proteins whichare candidate HLA class II binding peptides based on the consensus aminoacid sequences for binding HLA class II molecules. In this regard, see,e.g. International applications PCT/US96/03 182 and PCT/US98/01373.Peptides which are thus selected can be used in the assays describedherein for inducing specific CD4 lymphocytes and identification ofpeptides. Additional methods of selecting and testing peptides for HLAclass II binding are well known in the art.

[0055] As noted above, the invention embraces functional variants ofMAGE-A3 HLA class II binding peptides. As used herein, a “functionalvariant” or “variant” of a HLA class II binding peptide is a peptidewhich contains one or more modifications to the primary amino acidsequence of a HLA class II binding peptide and retains the HLA class IIand T cell receptor binding properties disclosed herein. Modificationswhich create a MAGE-A3 HLA class II binding peptide functional variantcan be made for example 1) to enhance a property of a MAGE-A3 HLA classII binding peptide, such as peptide stability in an expression system orthe stability of protein-protein binding such as HLA-peptide binding; 2)to provide a novel activity or property to a MAGE-A3 HLA class IIbinding peptide, such as addition of an antigenic epitope or addition ofa detectable moiety; or 3) to provide a different amino acid sequencethat produces the same or similar T cell stimulatory properties.Modifications to MAGE-A3 (as well as MAGE family) HLA class II bindingpeptides can be made to nucleic acids which encodes the peptide, and caninclude deletions, point mutations, truncations, amino acidsubstitutions and additions of amino acids. Alternatively, modificationscan be made directly to the polypeptide, such as by cleavage, additionof a linker molecule, addition of a detectable moiety, such as biotin,addition of a fatty acid, substitution of one amino acid for another andthe like. Variants also can be selected from libraries of peptides,which can be random peptides or peptides based on the sequence of theMAGE peptides including subtitutions at one or more positions. Forexample, a peptide library can be used in competition assays withcomplexes of MAGE peptides bound to HLA class II molecules (e.g.dendritic cells loaded with MAGE peptide). Peptides which compete forbinding of the MAGE peptide to the HLA class II molecule can besequenced and used in other assays (e.g. CD4 lymphocyte proliferation)to determine suitability as MAGE peptide functional variants.

[0056] Modifications also embrace fusion proteins comprising all or partof a MAGE HLA class II binding peptide amino acid sequence, such as theinvariant chain-MAGE-A3 fusion proteins described herein. The inventionthus embraces fusion proteins comprising MAGE-A3 HLA class II bindingpeptides and endosomal targeting signals such as the human invariantchain (Ii). As is disclosed below, fusion of an endosomal targetingportion of the human invariant chain to MAGE-A3 resulted in efficienttargeting of MAGE-A3 to the HLA class II peptide presentation pathway.An “endosomal targeting portion” of the human invariant chain or othertargeting polypeptide is that portion of the molecule which, when fusedor conjugated to a second polypeptide, increases endosomal localizationof the second polypeptide. Thus endosomal targeting portions can includethe entire sequence or only a small portion of a targeting polypeptidesuch as human invariant chain Ii. One of ordinary skill in the art canreadily determine an endosomal targeting portion of a targetingmolecule.

[0057] Prior investigations (PCT/US99/21230) noted that fusion of anendosomal targeting portion of LAMP-1 protein did not significantlyincrease targeting of MAGE-A3 to the HLA class II peptide presentationpathway. Therefore, the particular MAGE-A3 peptides of the invention canbe tested as fusions with LAMP-1 to determine if such fusion proteinsare efficiently targeted to the HLA class II peptide presentationpathway. Additional endosomal targeting signals can be identified by oneof ordinary skill in the art, fused to MAGE-A3 or a MAGE-A3 HLA class IIbinding portion thereof, and tested for targeting to the HLA class IIpeptide presentation pathway using no more than routine experimentation.

[0058] The amino acid sequence of MAGE HLA class II binding peptides maybe of natural or non-natural origin, that is, they may comprise anatural MAGE HLA class II binding peptide molecule or may comprise amodified sequence as long as the amino acid sequence retains the abilityto stimulate helper T cells when presented and retains the property ofbinding to an HLA class II molecule such as an HLA DRI molecule. Forexample, MAGE-A3 HLA class II binding peptides in this context may befusion proteins including a MAGE-A3 HLA class II binding peptide andunrelated amino acid sequences, synthetic MAGE-A3 HLA class II bindingpeptides, labeled peptides, peptides isolated from patients with aMAGE-A3 expressing cancer, peptides isolated from cultured cells whichexpress MAGE-A3, peptides coupled to nonpeptide molecules (for examplein certain drug delivery systems) and other molecules which include theamino acid sequence of SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:7.

[0059] Preferably, the MAGE-A3 HLA class II binding peptides arenon-hydrolyzable. To provide such peptides, one may select MAGE-A3 HLAclass II binding peptides from a library of non-hydrolyzable peptides,such as peptides containing one or more D-amino acids or peptidescontaining one or more non-hydrolyzable peptide bonds linking aminoacids. Alternatively, one can select peptides which are optimal forinducing CD4⁺ T lymphocytes and then modify such peptides as necessaryto reduce the potential for hydrolysis by proteases. For example, todetermine the susceptibility to proteolytic cleavage, peptides may belabeled and incubated with cell extracts or purified proteases and thenisolated to determine which peptide bonds are susceptible toproteolysis, e.g., by sequencing peptides and proteolytic fragments.Alternatively, potentially susceptible peptide bonds can be identifiedby comparing the amino acid sequence of a MAGE-A3 HLA class II bindingpeptide with the known cleavage site specificity of a panel ofproteases. Based on the results of such assays, individual peptide bondswhich are susceptible to proteolysis can be replaced withnon-hydrolyzable peptide bonds by in vitro synthesis of the peptide.

[0060] Many non-hydrolyzable peptide bonds are known in the art, alongwith procedures for synthesis of peptides containing such bonds.Non-hydrolyzable bonds include, but are not limited to,-psi[CH₂NH]-reduced amide peptide bonds, -psi[COCH₂]-ketomethylenepeptide bonds, -psi[CH(CN)NH]-(cyanomethylene)amino peptide bonds,-psi[CH₂CH(OH)]-hydroxyethylene peptide bonds, -psi[CH₂O]-peptide bonds,and -psi[CH₂S]-thiomethylene peptide bonds.

[0061] Nonpeptide analogs of peptides, e.g., those which provide astabilized structure or lessened biodegradation, are also contemplated.Peptide mimetic analogs can be prepared based on a selected MAGE-A3 HLAclass II binding peptide by replacement of one or more residues bynonpeptide moieties. Preferably, the nonpeptide moieties permit thepeptide to retain its natural conformation, or stabilize a preferred,e.g., bioactive, confirmation. Such peptides can be tested in molecularor cell-based binding assays to assess the effect of the substitution(s)on conformation and/or activity. One example of methods for preparationof nonpeptide mimetic analogs from peptides is described in Nachman etal., Regul. Pept. 57:359-370 (1995). Peptide as used herein embraces allof the foregoing.

[0062] If a variant involves a change to an amino acid of SEQ ID NO:3,SEQ ID NO:4, or SEQ ID NO:7, functional variants of the MAGE-A3 HLAclass II binding peptide having conservative amino acid substitutionstypically will be preferred, i.e., substitutions which retain a propertyof the original amino acid such as charge, hydrophobicity, conformation,etc. Examples of conservative substitutions of amino acids includesubstitutions made amongst amino acids within the following groups: (a)M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G; (e) S, T; (f) Q, N; and(g)E, D.

[0063] Other methods for identifying functional variants of the MAGE-A3HLA class II binding peptides are provided in a published PCTapplication of Strominger and Wucherpfennig (PCT/US96/03 182). Thesemethods rely upon the development of amino acid sequence motifs to whichpotential epitopes may be compared. Each motif describes a finite set ofamino acid sequences in which the residues at each (relative) positionmay be (a) restricted to a single residue, (b) allowed to vary amongst arestricted set of residues, or (c) allowed to vary amongst all possibleresidues. For example, a motif might specify that the residue at a firstposition may be any one of the residues valine, leucine, isoleucine,methionine, or phenylalanine; that the residue at the second positionmust be histidine; that the residue at the third position may be anyamino acid residue; that the residue at the fourth position may be anyone of the residues valine, leucine, isoleucine, methionine,phenylalanine, tyrosine or tryptophan; and that the residue at the fifthposition must be lysine.

[0064] Other computational methods for selecting amino acidsubstitutions, such as iterative computer structural modeling, can alsobe performed by one of ordinary skill in the art to prepare variants.Sequence motifs for MAGE-A3 HLA class II binding peptide functionalvariants can be developed by analysis of the binding domains or bindingpockets of major histocompatibility complex HLA-DR proteins and/or the Tcell receptor (“TCR”) contact points of the MAGE-A3 HLA class II bindingpeptides disclosed herein. By providing a detailed structural analysisof the residues involved in forming the HLA class II binding pockets,one is enabled to make predictions of sequence motifs for binding ofMAGE peptides to any of the HLA class II proteins.

[0065] Using these sequence motifs as search, evaluation, or designcriteria, one is enabled to identify classes of peptides (e.g. MAGE HLAclass II binding peptides, particularly the MAGE-A3 peptides disclosedherein, and functional variants thereof) which have a reasonablelikelihood of binding to a particular HLA molecule and of interactingwith a T cell receptor to induce T cell response. These peptides can besynthesized and tested for activity as described herein. Use of thesemotifs, as opposed to pure sequence homology (which excludes manypeptides which are antigenically similar but quite distinct in sequence)or sequence homology with unlimited “conservative” substitutions (whichadmits many peptides which differ at critical highly conserved sites),represents a method by which one of ordinary skill in the art canevaluate peptides for potential application in the treatment of disease.

[0066] The Strominger and Wucherpfennig PCT application, and referencescited therein, all of which are incorporated by reference, describe theHLA class II and TCR binding pockets which contact residues of an HLAclass II peptide. By keeping the residues which are likely to bind inthe HLA class II and/or TCR binding pockets constant or permitting onlyspecified substitutions, functional variants of MAGE HLA class IIbinding peptides can be prepared which retain binding to HLA class IIand T cell receptor.

[0067] Thus methods for identifying additional MAGE family HLA class IIpeptides, in particular MAGE-A3 HLA class II binding peptides, andfunctional variants thereof, are provided. In general, any MAGE proteincan be subjected to the analysis noted above, peptide sequences selectedand the tested as described herein. With respect to MAGE-A3, forexample, the methods include selecting a MAGE-A3 HLA class II bindingpeptide, an HLA class II binding molecule which binds the MAGE-A3 HLAclass II binding peptide, and a T cell which is stimulated by theMAGE-A3 HLA class II binding peptide presented by the HLA class IIbinding molecule. In preferred embodiments, the MAGE-A3 HLA class IIbinding peptide comprises the amino acid sequence of SEQ ID NO:3, SEQ IDNO:4 or SEQ ID NO:7. More preferably, the peptide consists essentiallyof those amino acid sequences. A first amino acid residue of the MAGE-A3HLA class II binding peptide is mutated to prepare a variant peptide.The amino acid residue can be mutated according to the principles of HLAand T cell receptor contact points set forth in the Strominger andWucherpfennig PCT application described above. Any method for preparingvariant peptides can be employed, such as synthesis of the variantpeptide, recombinantly producing the variant peptide using a mutatednucleic acid molecule, and the like.

[0068] The binding of the variant peptide to HLA class II bindingmolecule and stimulation of the T cell are then determined according tostandard procedures. For example, as exemplified below, the variantpeptide can be contacted with an antigen presenting cell which containsthe HLA class II molecule which binds the MAGE-A3 peptide to form acomplex of the variant peptide and antigen presenting cell. This complexcan then be contacted with a T cell which recognizes the MAGE-A3 HLAclass II binding peptide presented by the HLA class II binding molecule.T cells can be obtained from a patient having a condition characterizedby expression of MAGE-A3, such as cancer. Recognition of variantpeptides by the T cells can be determined by measuring an indicator of Tcell stimulation such as TNF IFNγ production. Similar procedures can becarried out for identification and characterization of other MAGE familyHLA class II binding peptides.

[0069] Binding of a variant peptide to the HLA class II binding moleculeand stimulation of the T cell by the variant peptide presented by theHLA class II binding molecule indicates that the variant peptide is afunctional variant. The methods also can include the step of comparingthe stimulation of the T cell by the MAGE-A3 HLA class II bindingpeptide and the stimulation of the T cell by the functional variant as adetermination of the effectiveness of the stimulation of the T cell bythe functional variant. By comparing the functional variant with theMAGE-A3 HLA class II binding peptide, peptides with increased T cellstimulatory proterties can be prepared.

[0070] The forgoing methods can be repeated sequentially with a second,third, fourth, fifth, sixth, seventh, eighth, ninth, and tenthsubstitutions to prepare additional functional variants of the disclosedMAGE-A3 HLA class II binding peptides.

[0071] Variants of the MAGE-A3 HLA class II binding peptides prepared byany of the foregoing methods can be sequenced, if necessary, todetermine the amino acid sequence and thus deduce the nucleotidesequence which encodes such variants.

[0072] Also a part of the invention are those nucleic acid sequenceswhich code for a MAGE HLA class II binding peptides or variants thereofand other nucleic acid sequences which hybridize to a nucleic acidmolecule consisting of the above described nucleotide sequences, understringent conditions. Preferred nucleic acid molecules include thosecomprising the nucleotide sequences that encode SEQ ID NOs:3, 4 and 7,which are SEQ ID NOs:5, 6 and 15, respectively. The term “stringentconditions” as used herein refers to parameters with which the art isfamiliar. Nucleic acid hybridization parameters may be found inreferences which compile such methods, e.g. Molecular Cloning: ALaboratory Manual, J. Sambrook, et al., eds., Second Edition, ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y. , 1989, orCurrent Protocols in Molecular Biology, F. M. Ausubel, et al., eds.,John Wiley & Sons, Inc., New York. More specifically, stringentconditions, as used herein, refers to hybridization at 65° C. inhybridization buffer (3.5× SSC, 0.02% Ficoll, 0.02% Polyvinylpyrolidone, 0.02% Bovine Serum Albumin, 25 mM NaH₂PO₄ (pH 7), 0.5% SDS,2 mM EDTA). SSC is 0.15M Sodium Chloride/0.15M Sodium Citrate, pH 7; SDSis Sodium Dodecyl Sulphate; and EDTA is Ethylene diaminetetraaceticacid. After hybridization, the membrane upon which the DNA istransferred is washed at 2× SSC at room temperature and then at 0.1-0.5×SSC/0.1× SDS at temperatures up to 68° C. Alternatively, stringenthybridization may be performed using a commercially availablehybridization buffer, such as ExpressHyb™ buffer (Clontech) usinghybridization and washing conditions described by the manufacturer.

[0073] There are other conditions, reagents, and so forth which canused, which result in a similar degree of stringency. The skilledartisan will be familiar with such conditions, and thus they are notgiven here. It will be understood, however, that the skilled artisanwill be able to manipulate the conditions in a manner to permit theclear identification of homologs and alleles of nucleic acids encodingthe MAGE HLA class II binding peptides of the invention. The skilledartisan also is familiar with the methodology for screening cells andlibraries for expression of such molecules which then are routinelyisolated, followed by isolation of the pertinent nucleic acid moleculeand sequencing.

[0074] In general homologs and alleles typically will share at least 90%amino acid identity and/or at least 75% nucleotide identity to the aminoacid sequence of a MAGE-A3 HLA class II binding peptide (such as SEQ IDNO:3, SEQ ID NO:4 and SEQ ID NO:7) or nucleic acids which encode such apeptide, respectively. In some instances homologs and alleles will shareat least 90% nucleotide identity and/or at least 95% amino acid identityand in still other instances will share at least 95% nucleotide identityand/or at least 99% amino acid identity. Complements of the foregoingnucleic acids also are embraced by the invention.

[0075] In screening for nucleic acids which encode a MAGE HLA class IIbinding peptide, a nucleic acid hybridization such as a Southern blot ora Northern blot may be performed using the foregoing conditions,together with a detectably labeled probe (e.g., radioactive such as ³²P,chemiluminescent, fluorescent labels). After washing the membrane towhich DNA encoding a MAGE HLA class II binding peptide was finallytransferred, the membrane can be placed against X-ray film,phosphorimager or other detection device to detect the detectable label.

[0076] The invention also includes the use of nucleic acid sequenceswhich include alternative codons that encode the same amino acidresidues of the MAGE HLA class II binding peptides. For example, asdisclosed herein, the peptide ACYEFLWGPRALVETS (SEQ ID NO:4) is aMAGE-A3 HLA class II binding peptide. The leucine residues (amino acidsNo. 6 and 12 of SEQ ID NO:4) can be encoded by the codons CUA, CUC, CUG,CUU, UUA and UUG. Each of the six codons is equivalent for the purposesof encoding a leucine residue. Thus, it will be apparent to one ofordinary skill in the art that any of the leucine-encoding nucleotidetriplets may be employed to direct the protein synthesis apparatus, invitro or in vivo, to incorporate a leucine residue. Similarly,nucleotide sequence triplets which encode other amino acid residuescomprising the MAGE-A3 HLA class II binding peptide of SEQ ID NO:4include: CGA, CGC, CGG, CGT, AGA and AGG (arginine codons); GUA, GUC,GUG and GUU (valine codons); GAA and GAG (glutamine codons); UUC and UUU(phenylalanine codons) and UAC and UAU (tyrosine codons). Other aminoacid residues may be encoded similarly by multiple nucleotide sequences.Thus, the invention embraces degenerate nucleic acids that differ fromthe native MAGE HLA class II binding peptide encoding nucleic acids incodon sequence due to the degeneracy of the genetic code.

[0077] It will also be understood that the invention embraces the use ofthe sequences in expression vectors, as well as to transfect host cellsand cell lines, be these prokaryotic (e.g., E. coli), or eukaryotic(e.g., dendritic cells, CHO cells, COS cells, yeast expression systemsand recombinant baculovirus expression in insect cells). The expressionvectors require that the pertinent sequence, i.e., those describedsupra, be operably linked to a promoter. As it has been found that humanHLA-DR1 molecules present a MAGE-A3 HLA class II binding peptide, theexpression vector may also include a nucleic acid sequence coding for anHLA-DR1 molecule. In a situation where the vector contains both codingsequences, it can be used to transfect a cell which does not normallyexpress either one. The MAGE-A3 HLA class II binding peptide codingsequence may be used alone, when, e.g. the host cell already expressesan HLA-DR1 molecule. Of course, there is no limit on the particular hostcell which can be used as the vectors which contain the two codingsequences may be used in host cells which do not express HLA-DR1molecules if desired, and the nucleic acid coding for the MAGE-A3 HLAclass H binding peptide can be used in antigen presenting cells whichexpress an HLA-DR1 molecule.

[0078] As used herein, “an HLA-DR1 molecule” includes the subtypesDRB1*0101, DRB1*01021, DRB1*01022, DRB1*0103, DRB1*0104, DRB1*0105,DRB1*0106, DRB1*0107 and other subtypes known to one of ordinary skillin the art. Other subtypes can be found in various publications thatupdate HLA allele lists.

[0079] As used herein, a “vector” may be any of a number of nucleicacids into which a desired sequence may be inserted by restriction andligation for transport between different genetic environments or forexpression in a host cell. Vectors are typically composed of DNAalthough RNA vectors are also available. Vectors include, but are notlimited to, plasmids, phagemids and virus genomes. A cloning vector isone which is able to replicate autonomously or after integration intothe genome in a host cell, and which is further characterized by one ormore endonuclease restriction sites at which the vector may be cut in adeterminable fashion and into which a desired DNA sequence may beligated such that the new recombinant vector retains its ability toreplicate in the host cell. In the case of plasmids, replication of thedesired sequence may occur many times as the plasmid increases in copynumber within the host bacterium or just a single time per host beforethe host reproduces by mitosis. In the case of phage, replication mayoccur actively during a lytic phase or passively during a lysogenicphase. An expression vector is one into which a desired DNA sequence maybe inserted by restriction and ligation such that it is operably joinedto regulatory sequences and may be expressed as an RNA transcript.Vectors may further contain one or more marker sequences suitable foruse in the identification of cells which have or have not beentransformed or transfected with the vector. Markers include, forexample, genes encoding proteins which increase or decrease eitherresistance or sensitivity to antibiotics or other compounds, genes whichencode enzymes whose activities are detectable by standard assays knownin the art (e.g., β-galactosidase, luciferase or alkaline phosphatase),and genes which visibly affect the phenotype of transformed ortransfected cells, hosts, colonies or plaques (e.g., green fluorescentprotein). Preferred vectors are those capable of autonomous replicationand expression of the structural gene products present in the DNAsegments to which they are operably joined.

[0080] Preferably the expression vectors contain sequences which targeta MAGE family polypeptide, e.g. MAGE-A3, or a HLA class II bindingpeptide derived therefrom, to the endosomes of a cell in which theprotein or peptide is expressed. HLA class II molecules contain aninvariant chain (Ii) which impedes binding of other molecules to the HLAclass II molecules. This invariant chain is cleaved in endosomes,thereby permitting binding of peptides by HLA class II molecules.Therefore it is preferable that the MAGE-A3 HLA class II bindingpeptides and precursors thereof (e.g. the MAGE-A3 protein) are targetedto the endosome, thereby enhancing MAGE-A3 HLA class II binding peptidebinding to HLA class II molecules. Targeting signals for directingmolecules to endosomes are known in the art and these signalsconveniently can be incorporated in expression vectors such that fusionproteins which contain the endosomal targeting signal are produced.Sanderson et al. (Proc. Nat'l. Acad. Sci. USA 92:7217-7221, 1995), Wu etal. (Proc. Nat'l. Acad. Sci. USA 92:11671-11675, 1995) and Thomson et al(J. Virol. 72:2246-2252, 1998) describe endosomal targeting signals(including invariant chain Ii and lysosomal-associated membrane proteinLAMP-1) and their use in directing antigens to endosomal and/orlysosomal cellular compartments. As disclosed in the Examples, invariantchain-MAGE-A3 fusion proteins are preferred.

[0081] Endosomal targeting signals such as invariant chain also can beconjugated to MAGE-A3 protein or peptides by non-peptide bonds (i.e. notfusion proteins) to prepare a conjugate capable of specificallytargeting MAGE-A3. Specific examples of covalent bonds include thosewherein bifunctional cross-linker molecules are used. The cross-linkermolecules may be homobifunctional or heterobifunctional, depending uponthe nature of the molecules to be conjugated. Homobifunctionalcross-linkers have two identical reactive groups. Heterobifunctionalcross-linkers are defined as having two different reactive groups thatallow for sequential conjugation reaction. Various types of commerciallyavailable cross-linkers are reactive with one or more of the followinggroups; primary amines, secondary amines, sulfhydryls, carboxyls,carbonyls and carbohydrates. One of ordinary skill in the art will beable to ascertain without undue experimentation the preferred moleculefor linking the endosomal targeting moiety and MAGE-A3 peptide orprotein, based on the chemical properties of the molecules being linkedand the preferred characteristics of the bond or bonds.

[0082] As used herein, a coding sequence and regulatory sequences aresaid to be “operably” joined when they are covalently linked in such away as to place the expression or transcription of the coding sequenceunder the influence or control of the regulatory sequences. If it isdesired that the coding sequences be translated into a functionalprotein, two DNA sequences are said to be operably joined if inductionof a promoter in the 5′ regulatory sequences results in thetranscription of the coding sequence and if the nature of the linkagebetween the two DNA sequences does not (1) result in the introduction ofa frame-shift mutation, (2) interfere with the ability of the promoterregion to direct the transcription of the coding sequences, or (3)interfere with the ability of the corresponding RNA transcript to betranslated into a protein. Thus, a promoter region would be operablyjoined to a coding sequence if the promoter region were capable ofeffecting transcription of that DNA sequence such that the resultingtranscript might be translated into the desired protein or polypeptide.

[0083] The precise nature of the regulatory sequences needed for geneexpression may vary between species or cell types, but shall in generalinclude, as necessary, 5 non-transcribed and 5′ non-translated sequencesinvolved with the initiation of transcription and translationrespectively, such as a TATA box, capping sequence, CAAT sequence, andthe like. In particular, such 5′ non-transcribed regulatory sequenceswill include a promoter region which includes a promoter sequence fortranscriptional control of the operably joined gene. Regulatorysequences may also include enhancer sequences or upstream activatorsequences as desired. The vectors of the invention may optionallyinclude 5′ leader or signal sequences. The choice and design of anappropriate vector is within the ability and discretion of one ofordinary skill in the art.

[0084] Expression vectors containing all the necessary elements forexpression are commercially available and known to those skilled in theart. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual,Second Edition, Cold Spring Harbor Laboratory Press, 1989. Cells aregenetically engineered by the introduction into the cells ofheterologous DNA (RNA) encoding a MAGE-A3 HLA class II binding peptide.That heterologous DNA (RNA) is placed under operable control oftranscriptional elements to permit the expression of the heterologousDNA in the host cell. As described herein, such expression constuctsoptionally also contain nucleotide sequences which encode endosomaltargeting signals, preferably human invariant chain or a targettingfragment thereof.

[0085] Preferred systems for mRNA expression in mammalian cells arethose such as pRc/CMV and pcDNA3.1 (available from Invitrogen, Carlsbad,Calif.) that contain a selectable marker such as a gene that confersG418 resistance (which facilitates the selection of stably transfectedcell lines) and the human cytomegalovirus (CMV) enhancer-promotersequences. Additionally, suitable for expression in primate or caninecell lines is the pCEP4 vector (Invitrogen), which contains an EpsteinBarr virus (EBV) origin of replication, facilitating the maintenance ofplasmid as a multicopy extrachromosomal element. Another expressionvector is the pEF-BOS plasmid containing the promoter of polypeptideElongation Factor 1α, which stimulates efficiently transcription invitro. The plasmid is described by Mishizuma and Nagata (Nuc. Acids Res.18:5322, 1990), and its use in transfection experiments is disclosed by,for example, Demoulin (Mol. Cell. Biol. 16:4710-4716, 1996). Stillanother preferred expression vector is an adenovirus, described byStratford-Perricaudet, which is defective for E1 and E3 proteins (J.Clin. Invest. 90:626-630, 1992). The use of the adenovirus as anAdeno.P1A recombinant is disclosed by Warnier et al., in intradermalinjection in mice for immunization against P1A (Int. J. Cancer,67:303-310, 1996). Recombinant vectors including viruses selected fromthe group consisting of adenoviruses, adeno-associated viruses,poxviruses including vaccinia viruses and attenuated poxviruses such asALVAC, NYVAC, Semliki Forest virus, Venezuelan equine encephalitisvirus, retroviruses, Sindbis virus, Ty virus-like particle, otheralphaviruses, VSV, plasmids (e.g. “naked” DNA), bacteria (e.g. thebacterium Bacille Calmette Guerin, attenuated Salmonella), and the likecan be used in such delivery, for example, for use as a vaccine.

[0086] The invention also embraces so-called expression kits, whichallow the artisan to prepare a desired expression vector or vectors.Such expression kits include at least separate portions of at least twoof the previously discussed materials. Other components may be added, asdesired.

[0087] The invention as described herein has a number of uses, some ofwhich are described herein. The following uses are described for MAGE-A3HLA class II binding peptides but are equally applicable to use of otherMAGE family HLA class II binding peptides. First, the invention permitsthe artisan to diagnose a disorder characterized by expression of aMAGE-A3 HLA class II binding peptide. These methods involve determiningexpression of a MAGE-A3 HLA class II binding peptide, or a complex of aMAGE-A3 HLA class II binding peptide and an HLA class II molecule in abiological sample. The expression of a peptide or complex of peptide andHLA class II molecule can be determined by assaying with a bindingpartner for the peptide or complex, such as an antibody.

[0088] The invention farther includes nucleic acid or proteinmicroarrays with components that bind MAGE-A3 HLA class II peptides ornucleic acids encoding such polypeptides. In this aspect of theinvention, standard techniques of microarray technology are utilized toassess expression of the MAGE-A3 polypeptides and/or identify biologicalconstituents that bind such polypeptides. The constituents of biologicalsamples include antibodies, lymphocytes (particularly T lymphocytes),and the like. Protein microarray technology, which is also known byother names including: protein chip technology and solid-phase proteinarray technology, is well known to those of ordinary skill in the artand is based on, but not limited to, obtaining an array of identifiedpeptides or proteins on a fixed substrate, binding target molecules orbiological constituents to the peptides, and evaluating such binding.See, e.g., G. MacBeath and S. L. Schreiber, “Printing Proteins asMicroarrays for High-Throughput Function Determination,” Science289(5485):1760-1763, 2000. Nucleic acid arrays, particularly arrays thatbind MAGE-A3 peptides also can be used for diagnostic applications, suchas for identifying subjects that have a condition characterized byMAGE-A3 polypeptide expression.

[0089] Microarray substrates include but are not limited to glass,silica, aluminosilicates, borosilicates, metal oxides such as aluminaand nickel oxide, various clays, nitrocellulose, or nylon. Themicroarray substrates may be coated with a compound to enhance synthesisof a probe (peptide or nucleic acid) on the substrate. Coupling agentsor groups on the substrate can be used to covalently link the firstnucleotide or amino acid to the substrate. A variety of coupling agentsor groups are known to those of skill in the art. Peptide or nucleicacid probes thus can be synthesized directly on the substrate in apredetermined grid. Alternatively, peptide or nucleic acid probes can bespotted on the substrate, and in such cases the substrate may be coatedwith a compound to enhance binding of the probe to the substrate. Inthese embodiments, presynthesized probes are applied to the substrate ina precise, predetermined volume and grid pattern, preferably utilizing acomputer-controlled robot to apply probe to the substrate in acontact-printing manner or in a non-contact manner such as ink jet orpiezo-electric delivery. Probes may be covalently linked to thesubstrate.

[0090] Targets are peptides or proteins and may be natural or synthetic.The tissue may be obtained from a subject or may be grown in culture(e.g. from a cell line).

[0091] In some embodiments of the invention one or more control peptideor protein molecules are attached to the substrate. Preferably, controlpeptide or protein molecules allow determination of factors such aspeptide or protein quality and binding characteristics, reagent qualityand effectiveness, binding success, and analysis thresholds and success.

[0092] In other embodiments, one or more control peptide or nucleic acidmolecules are attached to the substrate. Preferably, control nucleicacid molecules allow determination of factors such as bindingcharacteristics, reagent quality and effectiveness, hybridizationsuccess, and analysis thresholds and success.

[0093] Nucleic acid microarray technology, which is also known by othernames including: DNA chip technology, gene chip technology, andsolid-phase nucleic acid array technology, is well known to those ofordinary skill in the art and is based on, but not limited to, obtainingan array of identified nucleic acid probes on a fixed substrate,labeling target molecules with reporter molecules (e.g., radioactive,chemiluminescent, or fluorescent tags such as fluorescein, Cye3-dUTP, orCye5-dUTP), hybridizing target nucleic acids to the probes, andevaluating target-probe hybridization. A probe with a nucleic acidsequence that perfectly matches the target sequence will, in general,result in detection of a stronger reporter-molecule signal than willprobes with less perfect matches. Many components and techniquesutilized in nucleic acid microarray technology are presented in TheChipping Forecast, Nature Genetics, Vol.21, Jan 1999, the entirecontents of which is incorporated by reference herein.

[0094] According to the present invention, nucleic acid microarraysubstrates may include but are not limited to glass, silica,aluminosilicates, borosilicates, metal oxides such as alumina and nickeloxide, various clays, nitrocellulose, or nylon. In all embodiments aglass substrate is preferred. According to the invention, probes areselected from the group of nucleic acids including, but not limited to:DNA, genomic DNA, cDNA, and oligonucleotides; and may be natural orsynthetic. Oligonucleotide probes preferably are 20 to 25-meroligonucleotides and DNA/cDNA probes preferably are 500 to 5000 bases inlength, although other lengths may be used. Appropriate probe length maybe determined by one of ordinary skill in the art by following art-knownprocedures. In one embodiment, preferred probes are sets of two or moremolecule that bind the nucleic acid molecules that encode the MAGE-A3HLA class II binding peptides set forth herein. Probes may be purifiedto remove contaminants using standard methods known to those of ordinaryskill in the art such as gel filtration or precipitation.

[0095] In one embodiment, the microarray substrate may be coated with acompound to enhance synthesis of the probe on the substrate. Suchcompounds include, but are not limited to, oligoethylene glycols. Inanother embodiment, coupling agents or groups on the substrate can beused to covalently link the first nucleotide or olignucleotide to thesubstrate. These agents or groups may include, for example, amino,hydroxy, bromo, and carboxy groups. These reactive groups are preferablyattached to the substrate through a hydrocarbyl radical such as analkylene or phenylene divalent radical, one valence position occupied bythe chain bonding and the remaining attached to the reactive groups.These hydrocarbyl groups may contain up to about ten carbon atoms,preferably up to about six carbon atoms. Alkylene radicals are usuallypreferred containing two to four carbon atoms in the principal chain.These and additional details of the process are disclosed, for example,in U.S. Pat. No. 4,458,066, which is incorporated by reference in itsentirety.

[0096] In one embodiment, probes are synthesized directly on thesubstrate in a predetermined grid pattern using methods such aslight-directed chemical synthesis, photochemical deprotection, ordelivery of nucleotide precursors to the substrate and subsequent probeproduction.

[0097] In another embodiment, the substrate may be coated with acompound to enhance binding of the probe to the substrate. Suchcompounds include, but are not limited to: polylysine, amino silanes,amino-reactive silanes (Chipping Forecast, 1999) or chromium. In thisembodiment, presynthesized probes are applied to the substrate in aprecise, predetermined volume and grid pattern, utilizing acomputer-controlled robot to apply probe to the substrate in acontact-printing manner or in a non-contact manner such as ink jet orpiezo-electric delivery. Probes may be covalently linked to thesubstrate with methods that include, but are not limited to,UV-irradiation. In another embodiment probes are linked to the substratewith heat.

[0098] Targets for microarrays are nucleic acids selected from thegroup, including but not limited to: DNA, genomic DNA, cDNA, RNA, mRNAand may be natural or synthetic. In all embodiments, nucleic acid targetmolecules from human tissue are preferred. The tissue may be obtainedfrom a subject or may be grown in culture (e.g. from a cell line).

[0099] In embodiments of the invention one or more control nucleic acidmolecules are attached to the substrate. Preferably, control nucleicacid molecules allow determination of factors such as nucleic acidquality and binding characteristics, reagent quality and effectiveness,hybridization success, and analysis thresholds and success. Controlnucleic acids may include but are not limited to expression products ofgenes such as housekeeping genes or fragments thereof.

[0100] In some embodiments, one or more control peptide or nucleic acidmolecules are attached to the substrate. Preferably, control nucleicacid molecules allow determination of factors such as bindingcharacteristics, reagent quality and effectiveness, hybridizationsuccess, and analysis thresholds and success.

[0101] The invention also permits the artisan to treat a subject havinga disorder characterized by expression of a MAGE-A3 HLA class II bindingpeptide. Treatments include administering an agent which enriches in thesubject a complex of a MAGE-A3 HLA class II binding peptide and an HLAclass II molecule, and administering CD4⁺ T lymphocytes which arespecific for such complexes. Agents useful in the foregoing treatmentsinclude MAGE-A3 HLA class II binding peptides and functional variantsthereof, endosome-targeted fusion proteins which include such MAGE-A3peptides, nucleic acids which express such proteins and peptides(including viruses which contain the nucleic acids), complexes of suchpeptides and HLA class II binding molecules (e.g. HLA DR1), antigenpresenting cells bearing complexes of a MAGE-A3 HLA class II bindingpeptide and an HLA class II binding molecule, and the like. Theinvention also permits an artisan to selectively enrich a population ofT lymphocytes for CD4⁺ T lymphocytes specific for a MAGE-A3 HLA class IIbinding peptide.

[0102] The isolation of the MAGE-A3 HLA class II binding peptides alsomakes it possible to isolate nucleic acids which encode the MAGE-A3 HLAclass II binding peptides. Nucleic acids can be used to produce in vitroor in prokaryotic or eukaryotic host cells the MAGE-A3 HLA class IIbinding peptides. A variety of methodologies well-known to the skilledpractitioner can be utilized to obtain isolated MAGE-A3 HLA class IIbinding peptides. For example, an expression vector may be introducedinto cells to cause production of the peptides. In another method, mRNAtranscripts may be microinjected or otherwise introduced into cells tocause production of the encoded peptides. Translation of mRNA incell-free extracts such as the reticulocyte lysate system also may beused to produce peptides. Peptides comprising the MAGE-A3 HLA class IIbinding peptide of the invention may also be synthesized in vitro. Thoseskilled in the art also can readily follow known methods for isolatingpeptides in order to obtain isolated MAGE-A3 HLA class II bindingpeptides. These include, but are not limited to, immunochromatography,HPLC, size-exclusion chromatography, ion-exchange chromatography andimmune-affinity chromatography.

[0103] These isolated MAGE-A3 HLA class II binding peptides, proteinswhich include such peptides, or complexes of the peptides and HLA classII molecules, such as HLA-DR1 molecule, may be combined with materialssuch as adjuvants to produce vaccines useful in treating disorderscharacterized by expression of the MAGE-A3 HLA class II binding peptide.In addition, vaccines can be prepared from cells which present theMAGE-A3 HLA class II binding peptide/HLA complexes on their surface,such as dendritic cells, B cells, non-proliferative transfectants,etcetera. In all cases where cells are used as a vaccine, these can becells transfected with coding sequences for one or both of thecomponents necessary to stimulate CD4⁺ lymphocytes, or be cells whichalready express both molecules without the need for transfection. Forexample, autologous antigen presenting cells can be isolated from apatient and treated to obtain cells which present MAGE-A3 epitopes inassociation of HLA class I and HLA class II molecules. These cells wouldbe capable of stimulating both CD4⁺ and CD8⁺ cell responses. Suchantigen presenting cells can be obtained by infecting dendritic cellswith recombinant viruses encoding an Ii.MAGE-A3 fusion protein.Dendritic cells also can be loaded with HLA class I and HLA class IIepitopes.

[0104] Vaccines also encompass naked DNA or RNA, encoding a MAGE-A3 HLAclass II binding peptide or precursor thereof, which may be produced invitro and administered via injection, particle bombardment, nasalaspiration and other methods. Vaccines of the “naked nucleic acid” typehave been demonstrated to provoke an immunological response includinggeneration of CTLs specific for the peptide encoded by the naked nucleicacid (Science 259:1745-1748, 1993). Vaccines also include nucleic acidspackaged in a virus, liposome or other particle, including polymericparticles useful in drug delivery.

[0105] The immune response generated or enhanced by any of thetreatments described herein can be monitored by various methods known inthe art. For example, the presence of T cells specific for a givenantigen can be detected by direct labeling of T cell receptors withsoluble fluorogenic MHC molecule tetramers which present the antigenicpeptide (Altman et al., Science 274:94-96, 1996; Dunbar et al., Curr.Biol. 8:413-416, 1998). Briefly, soluble MHC class I molecules arefolded in vitro in the presence of β2-microglobulin and a peptideantigen which binds the class I molecule. After purification, theMHC/peptide complex is purified and labeled with biotin. Tetramers areformed by mixing the biotinylated peptide-MHC complex with labeledavidin (e.g. phycoerythrin) at a molar ratio of 4:1. Tetramers are thencontacted with a source of CTLs such as peripheral blood or lymph node.The tetramers bind CTLs which recognize the peptide antigen/MHC class Icomplex. Cells bound by the tetramers can be sorted by fluorescenceactivated cell sorting to isolate the reactive CTLs. The isolated CTLsthen can be expanded in vitro for use as described herein. The use ofMHC class II molecules as tetramers was recently demonstrated byCrawford et al. (Immunity 8:675-682, 1998). Multimeric soluble MHC classII molecules were complexed with a covalently attached peptide. Theclass II tetramers were shown to bind with appropriate specificity andaffinity to specific T cells. Thus tetramers can be used to monitor bothCD4⁺ and CD8⁺ cell responses to vaccination protocols.

[0106] The MAGE-A3 HLA class II binding peptide, as well as complexes ofMAGE-A3 HLA class II binding peptide and HLA molecule, also may be usedto produce antibodies, using standard techniques well known to the art.Standard reference works setting forth the general principles ofantibody production include Catty, D., Antibodies, A Practical Approach,Vol. 1, IRL Press, Washington D.C. (1988); Klein, J., Immunology: TheScience of Cell-Non-Cell Discrimination, John Wiley and Sons, New York(1982); Kennett, R., et al., Monoclonal Antibodies, Hybridoma, A NewDimension In Biological Analyses, Plenum Press, New York (1980);Campbell, A., Monoclonal Antibody Technology, in Laboratory Techniquesand Biochemistry and Molecular Biology, Vol. 13 (Burdon, R. et al.EDS.), Elsevier Amsterdam (1984); and Eisen, H. N., Microbiology, thirdedition, Davis, B. D. et al. EDS. (Harper & Rowe, Philadelphia (1980).

[0107] Significantly, as is well-known in the art, only a small portionof an antibody molecule, the paratope, is involved in the binding of theantibody to its epitope (see, in general, Clark, W.R. (1986) TheExperimental Foundations of Modem Immunology Wiley & Sons, Inc., NewYork; Roitt, I. (1991) Essential Immunology, 7th Ed., BlackwellScientific Publications, Oxford). The pFc′ and Fc regions, for example,are effectors of the complement cascade but are not involved in antigenbinding. An antibody from which the pFc′ region has been enzymaticallycleaved, or which has been produced without the pFc′ region, designatedan F(ab′)₂ fragment, retains both of the antigen binding sites of anintact antibody. Similarly, an antibody from which the Fc region hasbeen enzymatically cleaved, or which has been produced without the Fcregion, designated an Fab fragment, retains one of the antigen bindingsites of an intact antibody molecule. Proceeding further, Fab fragmentsconsist of a covalently bound antibody light chain and a portion of theantibody heavy chain denoted Fd. The Fd fragments are the majordeterminant of antibody specificity (a single Fd fragment may beassociated with up to ten different light chains without alteringantibody specificity) and Fd fragments retain epitope-binding ability inisolation.

[0108] Within the antigen-binding portion of an antibody, as iswell-known in the art, there are complementarity determining regions(CDRs), which directly interact with the epitope of the antigen, andframework regions (FRs), which maintain the tertiary structure of theparatope (see, in general, Clark, 1986; Roitt, 1991). In both the heavychain Fd fragment and the light chain of IgG immunoglobulins, there arefour framework regions (FR1 through FR4) separated respectively by threecomplementarity determining regions (CDR1 through CDR3). The CDRs, andin particular the CDR3 regions, and more particularly the heavy chainCDR3, are largely responsible for antibody specificity.

[0109] It is now well-established in the art that the non-CDR regions ofa mammalian antibody may be replaced with similar regions of conspecificor heterospecific antibodies while retaining the epitopic specificity ofthe original antibody. This is most clearly manifested in thedevelopment and use of “humanized” antibodies in which non-human CDRsare covalently joined to human FR and/or Fc/pFc′ regions to produce afunctional antibody. See, e.g., U.S. Pat. Nos. 4,816,567, 5,225,539,5,585,089, 5,693,762 and 5,859,205.

[0110] Fully human monoclonal antibodies also can be prepared byimmunizing mice transgenic for large portions of human immunoglobulinheavy and light chain loci. See, e.g., U.S. Pat. Nos. 5,545,806,6,150,584, and references cited therein. Following immunization of thesemice (e.g., XenoMouse (Abgenix), HuMAb mice (Medarex/GenPharm)),monoclonal antibodies can be prepared according to standard hybridomatechnology. These monoclonal antibodies will have human immunoglobulinamino acid sequences and therefore will not provoke human anti-mouseantibody (HAMA) responses when administered to humans.

[0111] Thus, as will be apparent to one of ordinary skill in the art,the present invention also provides for F(ab′)₂, Fab, Fv and Fdfragments; chimeric antibodies in which the Fc and/or FR and/or CDR1and/or CDR2 and/or light chain CDR3 regions have been replaced byhomologous human or non-human sequences; chimeric F(ab′)₂ fragmentantibodies in which the FR and/or CDR1 and/or CDR2 and/or light chainCDR3 regions have been replaced by homologous human or non-humansequences; chimeric Fab fragment antibodies in which the FR and/or CDR1and/or CDR2 and/or light chain CDR3 regions have been replaced byhomologous human or non-human sequences; and chimeric Fd fragmentantibodies in which the FR and/or CDR1 and/or CDR2 regions have beenreplaced by homologous human or non-human sequences. The presentinvention also includes so-called single chain antibodies.

[0112] The antibodies of the present invention thus are prepared by anyof a variety of methods, including administering protein, fragments ofprotein, cells expressing the protein or fragments thereof and anappropriate HLA class II molecule, and the like to an animal to inducepolyclonal antibodies. The production of monoclonal antibodies isaccording to techniques well known in the art. As detailed herein, suchantibodies may be used for example to identify tissues expressingprotein or to purify protein. Antibodies also may be coupled to specificlabeling agents for imaging or to antitumor agents, including, but notlimited to, methotrexate, radioiodinated compounds, toxins such asricin, other cytostatic or cytolytic drugs, and so forth. Antibodiesprepared according to the invention also preferably are specific for thepeptide/HLA complexes described herein.

[0113] When “disorder” or “condition” is used herein, it refers to anypathological condition where the MAGE-A3 HLA class II binding peptide isexpressed. Such disorders include cancers, such as melanomas, squamouscell carcinomas of the head, neck, lung or esophagus, colorectalcarcinomas, osteosarcomas, neuroblastomas, non-squamous cell carcinomasof the head or neck, ovarian tumors, lymphocytic leukemias, bladdercarcinomas, prostate carcinomas, etc.

[0114] Some therapeutic approaches based upon the disclosure arepremised on inducing a response by a subject's immune system to MAGE HLAclass II binding peptide presenting cells. One such approach is theadministration of autologous CD4⁺ T cells specific to the complex ofMAGE-A3 HLA class II binding peptide and an HLA class II molecule to asubject with abnormal cells of the phenotype at issue. It is within theskill of the artisan to develop such CD4⁺ T cells in vitro. Generally, asample of cells taken from a subject, such as blood cells, are contactedwith a cell presenting the complex and capable of provoking CD4⁺ Tlymphocytes to proliferate. The target cell can be a transfectant, suchas a COS cell, or an antigen presenting cell bearing HLA class IImolecules, such as dendritic cells or B cells. These transfectantspresent the desired complex of their surface and, when combined with aCD4⁺ T lymphocyte of interest, stimulate its proliferation. COS cellsare widely available, as are other suitable host cells. Specificproduction of CD4⁺ T lymphocytes is described below. The clonallyexpanded autologous CD4⁺ T lymphocytes then are administered to thesubject. The CD4⁺ T lymphocytes then stimulate the subject's immuneresponse, thereby achieving the desired therapeutic goal.

[0115] CTL proliferation can be increased by increasing the level oftryptophan in T cell cultures, by inhibiting enzymes which catabolizestryptophan, such as indoleamine 2,3-dioxygenase (IDO), or by addingtryptophan to the culture (see, e.g., PCT application WO99/293 10).Proliferation of T cells is enhanced by increasing the rate ofproliferation and/or extending the number of divisions of the T cells inculture. In addition, increasing tryptophan in T cell cultures alsoenhances the lytic activity of the T cells grown in culture.

[0116] The foregoing therapy assumes that at least some of the subject'sabnormal cells present the relevant HLA/peptide complex. This can bedetermined very easily, as the art is very familiar with methods foridentifying cells which present a particular HLA molecule, as well ashow to identify cells expressing DNA of the pertinent sequences, in thiscase a MAGE-A3 sequence.

[0117] The foregoing therapy is not the only form of therapy that isavailable in accordance with the invention. CD4⁺ T lymphocytes can alsobe provoked in vivo, using a number of approaches. One approach is theuse of non-proliferative cells expressing the complex. The cells used inthis approach may be those that normally express the complex, such asdendritic cells or cells transfected with one or both of the genesnecessary for presentation of the complex. Chen et al., (Proc. Natl.Acad. Sci. USA 88: 110-114, 1991) exemplifies this approach, showing theuse of transfected cells expressing HPV-E7 peptides in a therapeuticregime. Various cell types may be used. Similarly, vectors carrying oneor both of the genes of interest may be used. Viral or bacterial vectorsare especially preferred. For example, nucleic acids which encode aMAGE-A3 HLA class II binding peptide may be operably linked to promoterand enhancer sequences which direct expresion of the MAGE-A3 HLA classII binding peptide in certain tissues or cell types. The nucleic acidmay be incorporated into an expression vector. Expression vectors may beunmodified extrachromosomal nucleic acids, plasmids or viral genomesconstructed or modified to enable insertion of exogenous nucleic acids,such as those encoding MAGE-A3 HLA class II binding peptides. Nucleicacids encoding a MAGE-A3 HLA class II binding peptide also may beinserted into a retroviral genome, thereby facilitating integration ofthe nucleic acid into the genome of the target tissue or cell type. Inthese systems, the gene of interest is carried by a microorganism, e.g.,a Vaccinia virus, poxviruses in general, adenovirus, herpes simplexvirus, retrovirus or the bacteria BCG, and the materials de facto“infect” host cells. The cells which result present the complex ofinterest, and are recognized by autologous CD4⁺ T cells, which thenproliferate.

[0118] A similar effect can be achieved by combining a MAGE HLA class IIbinding peptide with an adjuvant to facilitate incorporation into HLAclass II presenting cells in vivo. If larger than the HLA class IIbinding portion, the MAGE-A3 HLA class II binding peptide can beprocessed if necessary to yield the peptide partner of the HLA moleculewhile the TRA is presented without the need for further processing.Generally, subjects can receive an intradermal injection of an effectiveamount of the MAGE-A3 HLA class II binding peptide. Initial doses can befollowed by booster doses, following immunization protocols standard inthe art.

[0119] A preferred method for facilitating incorporation of MAGE-A3 HLAclass II binding peptides into HLA class II presenting cells is byexpressing in the presenting cells a polypeptide which includes anendosomal targeting signal fused to a MAGE-A3 polypeptide which includesthe class II binding peptide. Particularly preferred are MAGE-A3 fusionproteins which contain human invariant chain Ii.

[0120] Any of the foregoing compositions or protocols can include alsoMAGE HLA class I binding peptides for induction of a cytolytic Tlymphocyte response. For example, as demonstrated below, the MAGE-A3protein can be processed in a cell to produce both HLA class I and HLAclass II responses. Several such peptides have been described in U.S.Pat. Nos. 5,585,461 and 5,591,430, and PCT published applicationPCT/US95/03657, as well as by Gaugler et al. (J. Exp. Med. 179:921-930,1994), van der Bruggen et al. (Eur. J. Immonol. 24:3038-3043, 1994), andHerman et al. (Immunogenetics 43:377-383, 1996). By administeringMAGE-A3 peptides which bind HLA class I and class II molecules (ornucleic acid encoding such peptides), an improved immune response may beprovided by inducing both T helper cells and T killer cells.

[0121] In addition, non-MAGE-A3 tumor associated peptides also can beadministered to increase immune response via HLA class I and/or classII. It is well established that cancer cells can express more that onetumor associated gene. It is within the scope of routine experimentationfor one of ordinary skill in the art to determine whether a particularsubject expresses additional tumor associated genes, and then includeHLA class I and/or HLA class II binding peptides derived from expressionproducts of such genes in the foregoing MAGE-A3 compositions andvaccines.

[0122] Especially preferred are nucleic acids encoding a series ofepitopes, known as “polytopes”. The epitopes can be arranged insequential or overlapping fashion (see, e.g., Thomson et al., Proc.Natl. Acad. Sci. USA 92:5845-5849, 1995; Gilbert et al., NatureBiotechnol. 15:1280-1284, 1997), with or without the natural flankingsequences, and can be separated by unrelated linker sequences ifdesired. The polytope is processed to generated individual epitopeswhich are recognized by the immune system for generation of immuneresponses.

[0123] Thus, for example, MAGE-A3 HLA class II binding peptides can becombined with peptides from other tumor rejection antigens (e.g. bypreparation of hybrid nucleic acids or polypeptides) and with MAGE-A3HLA class I binding peptides (some of which are listed below) to form“polytopes”. Exemplary tumor associated peptide antigens that can beadministered to induce or enhance an immune response are derived fromtumor associated genes and encoded proteins including MAGE-A1, MAGE-A2,MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10,MAGE-A11, MAGE-A12, MAGE-A13, GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5,GAGE-6, GAGE-7, GAGE-8, BAGE-1, RAGE-1, LB33/MUM-1, PRAME, NAG, MAGE-Xp2(MAGE-B2), MAGE-Xp3 (MAGE-B3), MAGE-Xp4 (MAGE-B4), tyrosinase, brainglycogen phosphorylase, Melan-A, MAGE-C1, MAGE-C2, NY-ESO-1, LAGE-1,SSX-1, SSX-2(HOM-MEL-40), SSX-1, SSX-4, SSX-5, SCP-1 and CT-7. Forexample, antigenic peptides characteristic of tumors include thoselisted in published PCT application WO 00/20581 (PCT/US99/21230).

[0124] Other examples of HLA class I and HLA class II binding peptideswill be known to one of ordinary skill in the art (for example, seeCoulie, Stem Cells 13:393-403, 1995), and can be used in the inventionin a like manner as those disclosed herein. One of ordinary skill in theart can prepare polypeptides comprising one or more MAGE-A3 peptides andone or more of the foregoing tumor rejection peptides, or nucleic acidsencoding such polypeptides, according to standard procedures ofmolecular biology.

[0125] Thus polytopes are groups of two or more potentially immunogenicor immune response stimulating peptides which can be joined together invarious arrangements (e.g. concatenated, overlapping). The polytope (ornucleic acid encoding the polytope) can be administered in a standardimmunization protocol, e.g. to animals, to test the effectiveness of thepolytope in stimulating, enhancing and/or provoking an immune response.

[0126] The peptides can be joined together directly or via the use offlanking sequences to form polytopes, and the use of polytopes asvaccines is well known in the art (see, e.g., Thomson et al., Proc.Acad. Natl. Acad. Sci USA 92(13):5845-5849, 1995; Gilbert et al., NatureBiotechnol. 15(12):1280-1284, 1997; Thomson et al., J. Immunol.157(2):822-826, 1996; Tam et al., J. Exp. Med. 171(1):299-306, 1990).For example, Tam showed that polytopes consisting of both MHC class Iand class II binding epitopes successfully generated antibody andprotective immunity in a mouse model. Tam also demonstrated thatpolytopes comprising “strings” of epitopes are processed to yieldindividual epitopes which are presented by MHC molecules and recognizedby CTLs. Thus polytopes containing various numbers and combinations ofepitopes can be prepared and tested for recognition by CTLs and forefficacy in increasing an immune response.

[0127] It is known that tumors express a set of tumor antigens, of whichonly certain subsets may be expressed in the tumor of any given patient.Polytopes can be prepared which correspond to the different combinationof epitopes representing the subset of tumor rejection antigensexpressed in a particular patient. Polytopes also can be prepared toreflect a broader spectrum of tumor rejection antigens known to beexpressed by a tumor type. Polytopes can be introduced to a patient inneed of such treatment as polypeptide structures, or via the use ofnucleic acid delivery systems known in the art (see, e.g., Allsopp etal., Eur. J. Immunol. 26(8):1951-1959, 1996). Adenovirus, pox virus,Ty-virus like particles, adeno-associated virus, plasmids, bacteria,etc. can be used in such delivery. One can test the polytope deliverysystems in mouse models to determine efficacy of the delivery system.The systems also can be tested in human clinical trials.

[0128] As part of the immunization compositions, one or more substancesthat potentiate an immune response are administered along with thepeptides described herein. Such substances include adjuvants andcytokines. An adjuvant is a substance incorporated into or administeredwith antigen which potentiates the immune response. Adjuvants mayenhance the immunological response by providing a reservoir of antigen(extracellularly or within macrophages), activating macrophages andstimulating specific sets of lymphocytes. Adjuvants of many kinds arewell known in the art. Specific examples of adjuvants includemonophosphoryl lipid A (MPL, SmithKline Beecham), a congener obtainedafter purification and acid hydrolysis of Salmonella Minnesota Re 595lipopolysaccharide; saponins including QS21 (SmithKline Beecham), a pureQA-21 saponin purified from Quillja saponaria extract; DQS21, describedin PCT application WO96/33739 (SmithKline Beecham); QS-7, QS-17, QS-18,and QS-L1 (So et al., Mol. Cells 7:178-186, 1997); incomplete Freund'sadjuvant; complete Freund's adjuvant; montanide; immunostimulatoryoligonucleotides (see e.g. CpG oligonucleotides described by Kreig etal., Nature 374:546-9, 1995); vitamin E and various water-in-oilemulsions prepared from biodegradable oils such as squalene and/ortocopherol. Preferably, the peptides are administered mixed with acombination of DQS21/MPL. The ratio of DQS21 to MPL typically will beabout 1:10 to 10:1, preferably about 1:5 to 5:1 and more preferablyabout 1:1. Typically for human administration, DQS21 and MPL will bepresent in a vaccine formulation in the range of about 1 μg to about 100μg. Other adjuvants are known in the art and can be used in theinvention (see, e.g. Goding, Monoclonal Antibodies: Principles andPractice, 2nd Ed., 1986). Methods for the preparation of mixtures oremulsions of peptide and adjuvant are well known to those of skill inthe art of vaccination.

[0129] Other agents which stimulate the immune response of the subjectcan also be administered to the subject. For example, other cytokinesare also useful in vaccination protocols as a result of their lymphocyteregulatory properties. Many other cytokines useful for such purposeswill be known to one of ordinary skill in the art, includinginterleukin-12 (IL-12) which has been shown to enhance the protectiveeffects of vaccines (see, e.g., Science 268:1432-1434, 1995), GM-CSF andIL-18. Thus cytokines can be administered in conjunction with antigensand adjuvants to increase the immune response to the antigens. There area number of additional immune response potentiating compounds that canbe used in vaccination protocols. These include costimulatory moleculesprovided in either protein or nucleic acid form. Such costimulatorymolecules include the B7-1 and B7-2 (CD80 and CD86 respectively)molecules which are expressed on dendritic cells (DC) and interact withthe CD28 molecule expressed on the T cell. This interaction providescostimulation (signal 2) to an antigen/MHC/TCR stimulated (signal 1) Tcell, increasing T cell proliferation and effector function. B7 alsointeracts with CTLA4 (CD152) on T cells and studies involving CTLA4 andB7 ligands indicate that the B7-CTLA4 interaction can enhance antitumorimmunity and CTL proliferation (Zheng et al., Proc. Nat'l Acad. Sci. USA95:6284-6289, 1998).

[0130] B7 typically is not expressed on tumor cells so they are notefficient antigen presenting cells (APCs) for T cells. Induction of B7expression would enable the tumor cells to stimulate more efficientlyCTL proliferation and effector function. A combination of B7/IL-6/IL-12costimulation has been shown to induce IFN-gamma and a Th1 cytokineprofile in the T cell population leading to further enhanced T cellactivity (Gajewski et al., J. Immunol. 154:5637-5648, 1995). Tumor celltransfection with B7 has been discussed in relation to in vitro CTLexpansion for adoptive transfer immunotherapy by Wang et al. (J.Immunother. 19:1-8, 1996). Other delivery mechanisms for the B7 moleculewould include nucleic acid (naked DNA) immunization (Kim et al., NatureBiotechnol. 15:7:641-646, 1997) and recombinant viruses such as adenoand pox (Wendtner et al., Gene Ther. 4:726-735, 1997). These systems areall amenable to the construction and use of expression cassettes for thecoexpression of B7 with other molecules of choice such as the antigensor fragment(s) of antigens discussed herein (including polytopes) orcytokines. These delivery systems can be used for induction of theappropriate molecules in vitro and for in vivo vaccination situations.The use of anti-CD28 antibodies to directly stimulate T cells in vitroand in vivo could also be considered. Similarly, the inducibleco-stimulatory molecule ICOS which induces T cell responses to foreignantigen could be modulated, for example, by use of anti-ICOS antibodies(Hutloff et al., Nature 397:263-266, 1999).

[0131] Lymphocyte function associated antigen-3 (LFA-3) is expressed onAPCs and some tumor cells and interacts with CD2 expressed on T cells.This interaction induces T cell IL-2 and IFN-gamma production and canthus complement but not substitute, the B7/CD28 costimulatoryinteraction (Parra et al., J. Immunol., 158:637-642, 1997; Fenton etal., J. Immunother., 21:95-108, 1998).

[0132] Lymphocyte function associated antigen-1 (LFA-1) is expressed onleukocytes and interacts with ICAM-1 expressed on APCs and some tumorcells. This interaction induces T cell IL-2 and IFN-gamma production andcan thus complement but not substitute, the B7/CD28 costimulatoryinteraction (Fenton et al., 1998). LFA-1 is thus a further example of acostimulatory molecule that could be provided in a vaccination protocolin the various ways discussed above for B7.

[0133] Complete CTL activation and effector function requires Th cellhelp through the interaction between the Th cell CD40L (CD40 ligand)molecule and the CD40 molecule expressed by DCs (Ridge et al., Nature393:474, 1998; Bennett et al., Nature 393:478, 1998; Schoenberger etal., Nature 393:480, 1998). This mechanism of this costimulatory signalis likely to involve upregulation of B7 and associated IL-6/IL-12production by the DC (APC). The CD40-CD40L interaction thus complementsthe signal 1 (antigen/MHC-TCR) and signal 2 (B7-CD28) interactions.

[0134] The use of anti-CD40 antibodies to stimulate DC cells directly,would be expected to enhance a response to tumor associated antigenswhich are normally encountered outside of an inflammatory context or arepresented by non-professional APCs (tumor cells). Other methods forinducing maturation of dendritic cells, e.g., by increasing CD40-CD40Linteraction, or by contacting DCs with CpG-containingoligodeoxynucleotides or stimulatory sugar moieties from extracellularmatrix, are known in the art. In these situations Th help and B7costimulation signals are not provided. This mechanism might be used inthe context of antigen pulsed DC based therapies or in situations whereTh epitopes have not been defined within known tumor associated antigenprecursors.

[0135] When administered, the therapeutic compositions of the presentinvention are administered in pharmaceutically acceptable preparations.Such preparations may routinely contain pharmaceutically acceptableconcentrations of salt, buffering agents, preservatives, compatiblecarriers, supplementary immune potentiating agents such as adjuvants andcytokines and optionally other therapeutic agents.

[0136] The preparations of the invention are administered in effectiveamounts. An effective amount is that amount of a pharmaceuticalpreparation that alone, or together with further doses, stimulates thedesired response. In the case of treating cancer, the desired responseis inhibiting the progression of the cancer. This may involve onlyslowing the progression of the disease temporarily, although morepreferably, it involves halting the progression of the diseasepermanently. In the case of inducing an immune response, the desiredresponse is an increase in antibodies or T lymphocytes which arespecific for the MAGE-A3 immunogen(s) employed. These desired responsescan be monitored by routine methods or can be monitored according todiagnostic methods of the invention discussed herein.

[0137] Where it is desired to stimulate an immune response using atherapeutic composition of the invention, this may involve thestimulation of a humoral antibody response resulting in an increase inantibody titer in serum, a clonal expansion of cytotoxic lymphocytes, orsome other desirable immunologic response. It is believed that doses ofimmunogens ranging from one nanogram/kilogram to 100 miligrams/kilogram,depending upon the mode of administration, would be effective. Thepreferred range is believed to be between 500 nanograms and 500micrograms per kilogram. The absolute amount will depend upon a varietyof factors, including the material selected for administration, whetherthe administration is in single or multiple doses, and individualpatient parameters including age, physical condition, size, weight, andthe stage of the disease. These factors are well known to those ofordinary skill in the art and can be addressed with no more than routineexperimentation.

EXAMPLES

[0138] Antigens encoded by MAGE-A3 and recognized by T cells areinteresting targets for tumor immunotherapy because they are strictlytumor-specific and shared by many tumors of various histological types.A number of MAGE-A3 antigenic peptides presented by HLA class Imolecules have been used in clinical trials and regressions of melanomametastasis have been observed. We report here the identification ofadditional MAGE-A3 epitopes, including ACYEFLWGPRALVETS (MAGE-A3₂₆₇₋₂₈₂;SEQ ID NO:4) and GSDPACYEFLWGPRAL (MAGE-A3₂₆₃₋₂₇₈; SEQ ID NO:3),presented to CD4⁺ T lymphocytes by HLA-DR1 molecules, which areexpressed in approximately 18% of Caucasians and 6% of Orientals. Thesenew epitopes may be useful both for therapeutic vaccination and for theevaluation of the immune response in cancer patients. To identify thepitopes, monocyte-derived dendritic cells from a cancer patient wereloaded with a recombinant MAGE-A3 protein and used to stimulateautologous CD4⁺ T cells. This patient had melanoma metastases expressingMAGE-A3 and was injected with a recombinant MAGE-A3 protein.

[0139] Materials and Methods

[0140] Cell Lines, Media, and Reagents.

[0141] The Epstein Barr Virus-transformed B (EBV-B) cell lines and thetumor cell lines MZ2-MEL43, NA41-MEL and SK37-MEL were cultured in IMDM(GIBCO BRL, Gaithersburg, Md.) supplemented with 10% fetal calf serum(GIBCO BRL), 0.24 mM L-asparagine, 0.55 mM L-arginine, 1.5 mML-glutamine (AAG), 100 U/ml penicillin and 100 μg/ml streptomycin. Humanrecombinant IL-2 was purchased from Eurocetus (Amsterdam, TheNetherlands), IL-7 from Genzyme (Cambridge, Mass.), GM-CSF from ScheringPlough (Brinny, England), TNF-α from R&D Systems (Abingdon, UnitedKingdom). Human recombinant IL-4, IL-6, and IL-12 were produced in ourlaboratory.

[0142] The PhoenixAMPHO cell line (kindly provided by Dr. Nolan,Stanford University School of Medicine, CA, USA) is a high titeramphotropic retrovirus producing cell line that has been generated bystable transfection of 293T cells with a Moloney GagPol-IRES-Lyt 2construct with an RSV promoter and a pPGK hygro selectable marker. Thesecells were then stably transfected with the Moloney amphotropic envelopegene driven by a CMV promoter and co-selected with the diphtheria toxinresistance gene (pHED-7). This producer cell line is helper virus free.

[0143] PhoenixAMPHO cells were cultured and passaged in 175 cm² flasksin DMEM (Life Technologies, Ghent, Belgium) supplemented with 10% heatinactivated FCS, 2 mM L-glutamine, 100 U/ml penicillin and 100 μg/mlstreptomycin.

[0144] MAGE-A3 Proteins.

[0145] Two different MAGE-A3 proteins were used. One was produced in ourlaboratory in Spodoptera frugiperda (Sf9) insect cells, hereafterreferred to as protein MAGE-A3^(insect). A baculovirus expression systemfrom PharMingen (San Diego, Calif.) was used. The coding sequence ofMAGE-A3 was cloned in pAcGP67-A (PharMingen), a baculovirus transfervector, downstream of the signal sequence of the gp67 surface protein ofAutographa californica nuclear polyhedrosis virus (AcNPV), a strain ofbaculovirus. For easier purification, a sequence encoding a histidinetail was added at the C-terminus of the sequence of MAGE-A3. DNA of therecombinant plasmid was co-transfected with DNA of lethally mutatedAcNPV into Sf9 insect cells. Co-transfection allows recombinationbetween homologous regions of the plasmid and the virus, transferringthe foreign gene from the vector to the AcNPV DNA.

[0146] Purification of the protein contained in the supernatant of Sf9insect cell cultures involved the following steps: anion exchange onDEAE-sephadex A-50 (Amersham-Pharmacia Biotech AB, Uppsala, Sweden),retention on HighTrap chelating column (Amersham-Pharmacia Biotech AB,Uppsala, Sweden), retention on HighTrap chelating column(Amersham-Pharmacia Biotech AB, Uppsala, Sweden) saturated with NiCl₂,affinity chromatography with immobilized antibody 57B (kindly providedby Dr. G. Spagnoli, Department of Surgery, Basel, Switzerland),concentration and dialysis. The purification of the MAGE-A3 protein wasmonitored using a particle counting immunoassay, where the latex beadswere coated with purified F(ab′)₂ obtained from a goat immunized againstMAGE-A3.

[0147] The other MAGE-A3 protein, hereafter referred to as proteinMAGE-A3^(bacteria), was produced in Escherichia coli bySmithKline-Beecham Biologicals (Rixensart, Belgium). MAGE-A3^(bacteria)was produced as a recombinant His-MAGE-A3 protein (MAGE-A3 with a Histag) or as a recombinant LipoD-MAGE-A3-His protein. LipoD-MAGE-A3-Hiscontains one third of the lipidic form of the Haemophilus influenzaeprotein at its N-terminal residue and a polyhistidine marker at itsC-terminal residue. The proteins were purified by standardchromatographic procedures.

[0148] Construction the Retrovirus Encoding Ii-MAGE-A3.

[0149] A recombinant retrovirus, pMFG-Ii80-MAGE-A3-(IRES)-ΔLNGFr wasconstructed (retro-Ii-MAGE). The sequence encoding a truncated form ofthe human low affinity nerve growth factor receptor (ΔLNGFr) wasamplified from plasmid pUC19-ΔLNGFr that was kindly provided by Dr. C.Traversari (Instituto Scientifico H. S. Raffaele, Milan, Italy). The PCRamplification was carried out using the following primers: sense:5′-CCCTCATGAGGGCAGGTGCCA (SEQ ID NO:17) CCG-3′ antisense:5′-CCCAGATCTCTAGAGGATTCC (SEQ ID NO:18) CCTGTTCCAC-3′

[0150] This PCR introduces a BspH1 site at the start codon and a Bgl2site downstream from the stop codon. A BamH1 site near the 3′ end wasdeleted. The PCR product was cloned in pCR2.1 and sequenced. The ΔLNGFrgene fragment was isolated from this vector as a BspH1-Not1 (from thepCR2.1 polylinker) fragment.

[0151] The IRES sequence derived from the encephalomyocarditis virus wastransferred from pGEM-EMC2 (kindly provided by Dr. J. -C. Renauld,Catholic University of Louvain, Brussels, Belgium) into pBluescript. AnEcoR1-Nco1 fragment from pBluescript-IRES was used for furtherconstruction.

[0152] Both the ΔNGFr and the IRES DNA fragments were ligated togetherinto pCR2. 1 EcoR1-Not1. This three fragment ligation resulted in aplasmid named pCR2.1-IRES-ΔLNGFr.

[0153] The IRES-ΔLNGFr sequence was then transferred into pMFG-Ii80,which encodes the first 80 amino acids of the human invariant chain(Ii80). A complete MAGE-A3 cDNA was then ligated downstream of Ii80 intopMFG-Ii80-(IRES)-ΔLNGFr to form pMFG-Ii80-MAGE-A3-(IRES)-ΔLNGFr,allowing the simultaneous expression of the Ii-MAGE-A3 fusion proteinand the ΔLNGF receptor. The procedure for transducing cell lines hasbeen described previously.

[0154] High titer MAGEA3-encoding recombinant retrovirus stocks weregenerated by introducing plasmid pMFG-Ii80-MAGE-A3-(IRES)-ΔLNGFr intoPhoenixAMPHO packaging cells by transfection, as described below.Retrovirus stocks were harvested and used for transduction as describedbelow.

[0155] Generation of High Titer MAGE-A3 Encoding Recombinant Retrovirus.

[0156] The MAGE-A3 encoding retroviral vector plasmidpMFG-Ii80-MAGE-A3-(IRES)-ΔLNGFr was introduced into the PhoenixAMPHOpackaging cells by transfection. The MFG retroviral vector is derivedfrom Moloney murine leukemia virus and is lacking in a drug resistancemarker nor does it express any other potential antigenic protein exceptfor the inserted cDNA (Rivière, Proc. Natl Acad. Sci. USA 92:6733-6737,1995). The transfection procedure is a modification of the calciumphosphate-mediated transfection protocol of Graham and van der Eb(Virology 54:536-539).

[0157] Twenty four hours prior to transfection, 10.8×10⁶ PhoenixAMPHOcells were plated in 14 ml cell growth medium in a 75 cm² tissue cultureflask (Falcon). After adding the cells, the flask was gently shakenforward and backward to distribute cells evenly about the flask bottom.The cells were incubated at 37° C. and 5% CO₂. At the time oftransfection, when the cells should have reached a confluence of 70-80%,the medium was removed and was replaced by 14 ml fresh PhoenixAMPHO cellgrowth medium containing 25 mM chloroquine (Sigma Chemical Co., St.Louis, Mo., USA). A transfection cocktail was prepared in a 50 ml tubeby adding 40 μg retroviral vector plasmid DNA to water and diluting to1575 μl final volume. To this DNA solution 225 μl of 2 M CaCl₂ (Sigma)was added. Then, 1800 μl of 2× HeBS (50 mM HEPES, 10 mM KCl, 12 mMdextrose, 280 mM NaCl and 1.5 mM Na₂HPO₄ dissolved in distilled water,filtered through 0.2 μ filter and stored at −20° C.) was added dropwiseto the DNA/CaCl₂ solution by vigorously bubbling for 15 seconds with anautomatic pipette. The DNA/CaCl₂/HeBS mix was added immediately anddropwise onto the cells and the flask was gently swirled to ensureuniform mixing of DNA/CaPO₄ particles. The cells were incubated at 37°C./5% CO₂ for 7 to 9 hours and the chloroquine containing medium waschanged for fresh PhoenixAMPHO cell growth medium. Approximately 24hours prior to the harvest of the retroviral supernatant, thePhoenixAMPHO medium was removed and gently replaced by 9 ml of EBV cellgrowth medium (Iscove's) containing only 2.5% FCS. The retroviralsupernatant was harvested 48 hours following transfection by removingthe medium from the cells and filtering through a 0.45 μ filter toremove cell debris. After harvest and filtration, the virus containingmedium was kept on ice, aliquoted in appropriate volumes in 15 mlpolypropylene tubes and stored at −80° C.

[0158] Retroviral Transduction of EBV Cell Lines.

[0159] The EBV transformed cells were infected by resuspending the cellsin an infection cocktail and centrifugation. Target cells wereresuspended in 60 mm tissue culture plates (Falcon) at a density of1.0×10⁶ cells in 4 ml infection cocktail. The plates were centrifugedfor 2 hours at 32° C. and 1200 rcf in an IEC centrifuge, rotor type 228.For each plate to be transduced, 4 ml of injection cocktail was preparedby diluting the viral supernatant 1:2 in EBV cell growth medium andadding protamine sulfate to a final concentration of 6 μg/ml.Centrifugation was followed by another 2 hours of incubation in ahumidified incubator at 37° C. and cells were transferred to 4 ml oftarget cell growth medium. This transduction cycle was carried outimmediately after plating the cells and was repeated at 24 and 48 hours.

[0160] Dendritic Cells and CD4⁺ Responder T Cells.

[0161] Blood cells were collected as buffy-coat preparations frommelanoma patient DDHK2, and processed essentially as describedpreviously in PCT/US99/21230. Briefly, peripheral blood mononuclearcells (PBMC) were isolated by centrifugation on Lymphoprep (NycomedPharma, Oslo, Norway). In order to minimize contamination of PBMC byplatelets, the preparation was first centrifuged for 20 min/1000 rpm atroom temperature. After removal of the top 20-25 ml, containing most ofthe platelets, the tubes were centrifuged for 20 min/1500 rpm at roomtemperature. PBMC were depleted of T cells by resetting with2-aminoethylisothiouronium (Sigma) treated sheep erythrocytes. Thelymphocyte-depleted PBMC were left to adhere for 2 hours at 37° C. inculture flasks (Falcon) at a density of 2×10⁶ cells/ml in RPMI 1640medium supplemented with L-asparagine (0.24 mM), L-arginine (0.55 mM),L-glutamine (1.5 mM) and 1% autologous serum (complete medium).Non-adherent cells were discarded.

[0162] Adherent cells (dendritic cells) were obtained by culturingmonocytes in the presence of IL-4 (200 U/ml) and GM-CSF (70 ng/ml) inRPMI 1640 medium supplemented with AAG and 1% autologous plasma. Onefourth of the medium was replaced by fresh medium and cytokines everytwo days. On day 7, the non-adherent cell population was used as asource of enriched dendritic cells. Rosetted T cells were treated withNH₄Cl (160 mM) to lyse the sheep erythrocytes, and washed. CD4⁺ Tlymphocytes were isolated from rosetted T cells by positive selectionusing an anti-CD4 monoclonal antibody coupled to magnetic microbeads(Miltenyi Biotech, Germany) and by sorting through a MACS, asrecommended by the manufacturer

[0163] Mixed Lymphocytes/Dendritic Cells Culture.

[0164] Dendritic cells (5×10⁵) were incubated at 37° C., 5% CO₂, for 20h in complete RPMI medium supplemented with IL-4, GM-CSF and TNF-α(5ng/ml) in the presence of MAGE-A3^(bacteria) (20 μg/ml). Cells werewashed and added at 10⁴ per round-bottomed microwell to 10⁵ CD4⁺ Tlymphocytes in 200 μl IMDM supplemented with AAG and 1% autologousplasma in the presence of IL-6 (1,000 U/ml) and IL-12 (10 ng/ml). TheCD4⁺ lymphocytes were restimulated on days 7, 14, 21 and 28 withautologous dendritic cells freshly loaded with MAGE-A3^(bacteria) andgrown in IMDM supplemented with AAG and 1% autologous plasma (hereafterreferred to as complete IMDM) supplemented with IL-2 (10 U/ml) and IL-7(5 ng/ml). Aliquots of each microculture (˜5,000 cells) were assessed ondays 35 and 42 for their capacity to produce IFN-γ when stimulated with˜20,000 autologous EBV-B cells loaded for 20 h with either 20 μg/ml ofMAGE-A3^(bacteria), MAGE-A3^(insect), or ovalbumin. After 20 h ofco-culture in round-bottom microwells and in 100 μl complete IMDM mediumsupplemented with IL-2 (25 U/ml), IFN-γ released in the supernatant wasmeasured by ELISA using reagents from Medgenix Diagnostics-Biosource(Fleurus, Belgium).

[0165] CD4⁺ T Cell Clones.

[0166] Cells from positive microculture F4 that were cloned by limitingdilution, using irradiated autologous EBV-B cells transduced withretro-Ii.MAGE-A3 (5×10³-2×10⁴ cells) as stimulator cells. Irradiatedallogeneic LG2-EBV cells (5×10³-10⁴) were used as feeder cells. CD4⁺ Tcell clones were supplemented once a week with fresh culture medium inthe presence of IL-2 (50 U/ml), L-7 (5 ng/ml) and IL-4 (5 U/ml).

[0167] Recognition Assays with Peptides.

[0168] Peptides were synthesized on solid phase using F-moc fortransient NH2-terminal protection and were characterized using massspectrometry. All peptides were >90% pure, as indicated by analyticalHPLC. Lyophilized peptides were dissolved at 5 mg/ml in 10 mM aceticacid and 10% DMSO, and stored at −20° C. EBV-B cells were distributed at20,000 cells per round-bottomed microwell and incubated for 2 h at 37°C. in the presence of the different peptides, the indicatedconcentrations representing their concentrations during the incubationstep. CD4⁺ T lymphocytes (5,000) were added in 100 μl of complete IMDM(GIBCO) medium supplemented with IL-2 (25 U/ml). Supernatants wereharvested after 20 h of co-culture and IFN-y production was measured byELISA.

[0169] Recognition of Tumor Cells.

[0170] Tumor cells were distributed at 20,000 cells per round-bottomedmicrowell together with 5,000 CD4⁺ T lymphocytes in 100 μl of completeIMDM to medium in the presence of IL-2 (25 U/ml). Supernatants wereharvested after 20 h of co-culture and the IFN-γ production was measuredby ELISA.

[0171] Results

[0172] To identify new MAGE-A3 epitopes presented by HLA class IImolecules, monocyte-derived dendritic cells (DC) from melanoma patientDDHK2 were cultured in autologous plasma and incubated overnight with arecombinant MAGE-A3 protein and with TNF-α to induce their maturation.These cells were then used to stimulate autologous CD4⁺ T lymphocytes.In previous experiments, a large number of the CD4⁺ T cell clonesobtained with the same method were apparently directed against bacterialcontaminants in the batch of protein. Therefore, a MAGE-A3 proteinproduced in Escherichia coli (MAGE-A3^(bacteria)) was used to stimulatethe lymphocytes, and a MAGE-A3 protein produced in Spodoptera frugiperdainsect cells (MAGE-A3^(insect)) to test the specificity of the responderlymphoctyes (data not shown).

[0173] A CD4⁺ T Cell Clone Directed Against a MAGE-A3 Antigen.

[0174] A total of 96 microcultures were set up, each containing 10⁵ CD4⁺cells and 10⁴ autologous stimulator DC loaded with proteinMAGE-A3^(bacteria) as stimulator cells. Responder cells wererestimulated three times at weekly intervals with DC loaded with theprotein. After a resting period of two weeks, responder cells of eachmicroculture were tested on days 35 and 42 for IFN-γ production afterstimulation with autologous EBV-B cells loaded with eitherMAGE-A3^(insect), MAGE-A3^(bacteria) or ovalbumin. Twelve microculturesspecifically produced a high level of IFN-γ after stimulation withprotein MAGE-A3^(insect) and protein MAGE-A3^(bacteria). One of them,F4, was cloned by limiting dilution using autologous EBV-B cellstransduced with retro-Ii.MAGE-A3 as stimulator cells. Several positiveclones were obtained, including CD4⁺ T cell clone MAGJ569/F4.3. Thisclone recognized autologous EBV-B cells loaded with either proteinMAGE-A3^(insect) or protein MAGE-A3^(bacteria), or EBV-B cellstransduced with retro-Ii.MAGE-A3.

[0175] Autologous DDHK2-EBV-B cells were pulsed for 20 h with 20 μg/mlof protein MAGE-A3^(insect) or protein MAGE-A3^(bacteria).Protein-pulsed EBV-B cells (20,000) or EBV-B cells transduced with aretrovirus encoding a fusion protein composed of MAGE-A3 and a truncatedhuman invariant chain (retro-Ii.MAGE-A3) were incubated for 20 h inmicrowells with CD4⁺ clone MAGJ569/F4.3 cells (5,000). IFN-γ productionwas measured by ELISA (FIG. 1). The results shown represent the averageof triplicate cultures.

[0176] Clone MAGJ569/F4.3 Recognized Peptide ACYEFLWGPRALVETS (SEQ IDNO:4).

[0177] A set of peptides of 16 amino acids, which overlapped by 12 andcovered the entire MAGE-A3 protein sequence, was screened: autologousEBV-B cells were pulsed with each of these peptides and tested forrecognition by clone MAGJ569/F4.3. It produced IFN-γ after stimulationwith two overlapping peptides, namely MAGE-A3₂₆₃₋₂₇₈ (GSDPACYEFLWGPRAL;SEQ ID NO:3) and MAGE-A3₂₆₇₋₂₈₂ (ACYEFLWGPRALVETS; SEQ ID NO:4).

[0178] DDHK EBV-B cells (20,000) were incubated in microwells for 2hours with different concentrations of the MAGE-A3 peptides. IFN-γproduction was measured by ELISA after 20 hours of coculture with CD4⁺clone MAGJ569/F4.3 (5,000 cells) (FIG. 2). The experiments wereperformed twice.

[0179]FIG. 2A shows Experiment I. A number of MAGE-A3 peptides ofdifferent lengths were tested and recognized by clone MAGJ569/F4.3. Thepeptides tested were GSDPACYEFLWGPRAL (MAGE-A3₂₆₃₋₂₇₈; SEQ ID NO:3),ACYEFLWGPRALVETS (MAGE-A3₂₆₇₋₂₈₂; SEQ ID NO:4), ACYEFLWGPRALVE (SEQ IDNO:7), ACYEFLWGPRALV (SEQ ID NO:8), ACYEFLWGPRAL (SEQ ID NO:9),ACYEFLWGPRA (SEQ ID NO:10), ACYEFLWGPR (SEQ ID NO:11), CYEFLWGPRALVE(SEQ ID NO: 12), YEFLWGPRALVE (SEQ ID NO: 13), and EFLWGPRALVE (SEQ IDNO:14). Of these, GSDPACYEFLWGPRAL (SEQ ID NO:3), ACYEFLWGPRALVETS (SEQID NO:4), and ACYEFLWGPRALVE (SEQ ID NO:7) were well recognized. It isexpected that ACYEFLWGPRALVET (SEQ ID NO:16), as intermediate betweenthe amino acid sequences of SEQ ID NO:4 and SEQ ID NO:7, also would bewell recognized.

[0180]FIG. 2B shows Experiment II. For this second experiment,concentration of peptides were measured by optical density. The peptidestested were GSDPACYEFLWGPRAL (SEQ ID NO:3), ACYEFLWGPRALVETS (SEQ IDNO:4), ACYEFLWGPRALVE (SEQ ID NO:7) and ACYEFLWGPRALV (SEQ ID NO:8). Ofthese, GSDPACYEFLWGPRAL (SEQ ID NO:3), ACYEFLWGPRALVETS (SEQ ID NO:4),and ACYEFLWGPRALVE (SEQ ID NO:7) were well recognized, whileACYEFLWGPRALV (SEQ ID NO:8) was recognized but only at higherconcentrations of peptide.

[0181] The peptide is Presented by HLA-DR1 Molecules.

[0182] DDHK2-EBV transduced with retro-Ii.MAGE-A3 (20,000) werecocultured for 20 hours with CD4⁺ clone MAGJ569/F4.3 cells (5,000), inthe presence of either monoclonal antibody B7.21 (anti-DP), SPV L3(anti-DQ), or AH89 (anti-DR). IFN-γ production was measured by ELISA.The recognition by clone MAGJ569/F4.3 of autologous EBV-B cells loadedwith peptide MAGE-A3₂₆₇₋₂₈₂ (ACYEFLWGPRALVETS; SEQ ID NO:4) wasabolished by an anti-HLA-DR antibody, but not by antibodies againstHLA-DP or HLA-DQ (FIG. 3).

[0183] Melanoma patient DDHK2 was typed HLA-DR1, DR15 and DR51. PeptideACYEFLWGPRALVETS (SEQ ID NO:4) was loaded on several EBV-B cell linesexpressing DR1, DR15 or DR51. All and only those expressing DR1 wereable to present the peptide to clone MAGJ569/F4.3 (Table 1). AutologousDDHK2-EBV and allogeneic EBV-B cells (20,000) were incubated for 1 hourwith 5 μg/ml of peptide ACYEFLWGPRALVETS (MAGE-A3₂₆₇₋₂₈₂; SEQ ID NO:4)and washed. Peptide-pulsed EBV-B cells (20,000) were then incubated withCD4⁺ clone MAGJ565/F4.3 cells (5,000). IFN-γ production was measured byELISA after 20 hours of co-culture. TABLE 1 Presentation of the MAGE-A3peptide (MAGE362) by HLA-DR1 cells EBV-B cell line Serologicalspecificity IFN-γ production (pg/ml) DR1 positive DDHK2 DR1 DR15DR51 >4000 LB1158 DR1 DR13 DR52 2439 LB831 DR1 DR7 DR53 2037 LB2138 DR1DR13 1810 DR1 negative LB650 DR7 DR15 DR51 DR53 83 LB1870 DR15 DR53 DR788 LB1856 DR15 49 LB2095 DR13 DR15 DR51 DR52 22

[0184] Recognition of tumor cell lines. Three DR1 melanoma cell linesexpressing MAGE-A3 were tested for their ability to stimulate CD4⁺ Tcell clone MAGJ569/F4.3 to produce IFN-γ. Tumor cells were pretreatedfor 48 h with 100 U/ml of IFN-γ and were pulsed in microwells (20,000)for 1 h with 1 μg/ml of peptide ACYEFLWGPRALVETS (MAGE-A3₂₆₇₋₂₈₂; SEQ IDNO:4). IFN-γproduction was measured by ELISA after 20 h of coculturewith CD4+clone MAGJ610/F4.3 (5,000). Autologous EBV-stimulator cellstransduced with a retrovirus encoding Ii.MAGE-A3 were used as positivecontrols.

[0185] One of the melanoma cell lines, NA41-MEL, slightly stimulated theCD4⁺ clone MAGJ569/F4.3 to produce IFN-γ (FIG. 4). Treatment of thiscell line with IFN-γ improved its recognition.

[0186] Other aspects of the invention will be clear to the skilledartisan and need not be repeated here. Each reference cited herein isincorporated by reference in its entirety.

[0187] The terms and expressions which have been employed are used asterms of description and not of limitation, and there is no intention inthe use of such terms and expressions of excluding any equivalents ofthe features shown and described or portions thereof, it beingrecognized that various modifications are possible within the scope ofthe invention.

1 18 1 4204 DNA Homo sapiens CDS (2465)..(3409) 1 acgcaggcag tgatgtcacccagaccacac cccttccccc aatgccactt cagggggtac 60 tcagagtcag agacttggtctgaggggagc agaagcaatc tgcagaggat ggcggtccag 120 gctcagccag gcatcaacttcaggaccctg agggatgacc gaaggccccg cccacccacc 180 cccaactccc ccgaccccaccaggatctac agcctcagga cccccgtccc aatccttacc 240 ccttgcccca tcaccatcttcatgcttacc tccaccccca tccgatcccc atccaggcag 300 aatccagttc cacccctgcccggaacccag ggtagtaccg ttgccaggat gtgacgccac 360 tgacttgcgc attggaggtcagaagaccgc gagattctcg ccctgagcaa cgagcgacgg 420 cctgacgtcg gcggagggaagccggcccag gctcggtgag gaggcaaggt aagacgctga 480 gggaggactg aggcgggcctcacctcagac agagggcctc aaataatcca gtgctgcctc 540 tgctgccggg cctgggccaccccgcagggg aagacttcca ggctgggtcg ccactacctc 600 accccgccga cccccgccgctttagccacg gggaactctg gggacagagc ttaatgtggc 660 cagggcaggg ctggttagaagaggtcaggg cccacgctgt ggcaggaatc aaggtcagga 720 ccccgagagg gaactgagggcagcctaacc accaccctca ccaccattcc cgtcccccaa 780 cacccaaccc cacccccatcccccattccc atccccaccc ccacccctat cctggcagaa 840 tccgggcttt gcccctggtatcaagtcacg gaagctccgg gaatggcggc caggcacgtg 900 agtcctgagg ttcacatctacggctaaggg agggaagggg ttcggtatcg cgagtatggc 960 cgttgggagg cagcgaaagggcccaggcct cctggaagac agtggagtcc tgaggggacc 1020 cagcatgcca ggacagggggcccactgtac ccctgtctca aaccgaggca ccttttcatt 1080 cggctacggg aatcctagggatgcagaccc acttcagcag ggggttgggg cccagccctg 1140 cgaggagtca tggggaggaagaagagggag gactgagggg accttggagt ccagatcagt 1200 ggcaaccttg ggctgggggatgctgggcac agtggccaaa tgtgctctgt gctcattgcg 1260 ccttcagggt gaccagagagttgagggctg tggtctgaag agtgggactt caggtcagca 1320 gagggaggaa tcccaggatctgcagggccc aaggtgtacc cccaaggggc ccctatgtgg 1380 tggacagatg cagtggtcctaggatctgcc aagcatccag gtgaagagac tgagggagga 1440 ttgagggtac ccctgggacagaatgcggac tgggggcccc ataaaaatct gccctgctcc 1500 tgctgttacc tcagagagcctgggcagggc tgtcagctga ggtccctcca ttatcctagg 1560 atcactgatg tcagggaaggggaagccttg gtctgagggg gctgcactca gggcagtaga 1620 gggaggctct cagaccctactaggagtgga ggtgaggacc aagcagtctc ctcacccagg 1680 gtacatggac ttcaataaatttggacatct ctcgttgtcc tttccgggag gacctgggaa 1740 tgtatggcca gatgtgggtcccctcatgtt tttctgtacc atatcaggta tgtgagttct 1800 tgacatgaga gattctcaggccagcagaag ggagggatta ggccctataa ggagaaaggt 1860 gagggccctg agtgagcacagaggggatcc tccaccccag tagagtgggg acctcacaga 1920 gtctggccaa ccctcctgacagttctggga atccgtggct gcgtttgctg tctgcacatt 1980 gggggcccgt ggattcctctcccaggaatc aggagctcca ggaacaaggc agtgaggact 2040 tggtctgagg cagtgtcctcaggtcacaga gtagaggggg ctcagatagt gccaacggtg 2100 aaggtttgcc ttggattcaaaccaagggcc ccacctgccc cagaacacat ggactccaga 2160 gcgcctggcc tcaccctcaatactttcagt cctgcagcct cagcatgcgc tggccggatg 2220 taccctgagg tgccctctcacttcctcctt caggttctga ggggacaggc tgacctggag 2280 gaccagaggc ccccggaggagcactgaagg agaagatctg taagtaagcc tttgttagag 2340 cctccaaggt tccattcagtactcagctga ggtctctcac atgctccctc tctccccagg 2400 ccagtgggtc tccattgcccagctcctgcc cacactcccg cctgttgccc tgaccagagt 2460 catc atg cct ctt gagcag agg agt cag cac tgc aag cct gaa gaa ggc 2509 Met Pro Leu Glu Gln ArgSer Gln His Cys Lys Pro Glu Glu Gly 1 5 10 15 ctt gag gcc cga gga gaggcc ctg ggc ctg gtg ggt gcg cag gct cct 2557 Leu Glu Ala Arg Gly Glu AlaLeu Gly Leu Val Gly Ala Gln Ala Pro 20 25 30 gct act gag gag cag gag gctgcc tcc tcc tct tct act cta gtt gaa 2605 Ala Thr Glu Glu Gln Glu Ala AlaSer Ser Ser Ser Thr Leu Val Glu 35 40 45 gtc acc ctg ggg gag gtg cct gctgcc gag tca cca gat cct ccc cag 2653 Val Thr Leu Gly Glu Val Pro Ala AlaGlu Ser Pro Asp Pro Pro Gln 50 55 60 agt cct cag gga gcc tcc agc ctc cccact acc atg aac tac cct ctc 2701 Ser Pro Gln Gly Ala Ser Ser Leu Pro ThrThr Met Asn Tyr Pro Leu 65 70 75 tgg agc caa tcc tat gag gac tcc agc aaccaa gaa gag gag ggg cca 2749 Trp Ser Gln Ser Tyr Glu Asp Ser Ser Asn GlnGlu Glu Glu Gly Pro 80 85 90 95 agc acc ttc cct gac ctg gag tcc gag ttccaa gca gca ctc agt agg 2797 Ser Thr Phe Pro Asp Leu Glu Ser Glu Phe GlnAla Ala Leu Ser Arg 100 105 110 aag gtg gcc gag ttg gtt cat ttt ctg ctcctc aag tat cga gcc agg 2845 Lys Val Ala Glu Leu Val His Phe Leu Leu LeuLys Tyr Arg Ala Arg 115 120 125 gag ccg gtc aca aag gca gaa atg ctg gggagt gtc gtc gga aat tgg 2893 Glu Pro Val Thr Lys Ala Glu Met Leu Gly SerVal Val Gly Asn Trp 130 135 140 cag tat ttc ttt cct gtg atc ttc agc aaagct tcc agt tcc ttg cag 2941 Gln Tyr Phe Phe Pro Val Ile Phe Ser Lys AlaSer Ser Ser Leu Gln 145 150 155 ctg gtc ttt ggc atc gag ctg atg gaa gtggac ccc atc ggc cac ttg 2989 Leu Val Phe Gly Ile Glu Leu Met Glu Val AspPro Ile Gly His Leu 160 165 170 175 tac atc ttt gcc acc tgc ctg ggc ctctcc tac gat ggc ctg ctg ggt 3037 Tyr Ile Phe Ala Thr Cys Leu Gly Leu SerTyr Asp Gly Leu Leu Gly 180 185 190 gac aat cag atc atg ccc aag gca ggcctc ctg ata atc gtc ctg gcc 3085 Asp Asn Gln Ile Met Pro Lys Ala Gly LeuLeu Ile Ile Val Leu Ala 195 200 205 ata atc gca aga gag ggc gac tgt gcccct gag gag aaa atc tgg gag 3133 Ile Ile Ala Arg Glu Gly Asp Cys Ala ProGlu Glu Lys Ile Trp Glu 210 215 220 gag ctg agt gtg tta gag gtg ttt gagggg agg gaa gac agt atc ttg 3181 Glu Leu Ser Val Leu Glu Val Phe Glu GlyArg Glu Asp Ser Ile Leu 225 230 235 ggg gat ccc aag aag ctg ctc acc caacat ttc gtg cag gaa aac tac 3229 Gly Asp Pro Lys Lys Leu Leu Thr Gln HisPhe Val Gln Glu Asn Tyr 240 245 250 255 ctg gag tac cgg cag gtc ccc ggcagt gat cct gca tgt tat gaa ttc 3277 Leu Glu Tyr Arg Gln Val Pro Gly SerAsp Pro Ala Cys Tyr Glu Phe 260 265 270 ctg tgg ggt cca agg gcc ctc gttgaa acc agc tat gtg aaa gtc ctg 3325 Leu Trp Gly Pro Arg Ala Leu Val GluThr Ser Tyr Val Lys Val Leu 275 280 285 cac cat atg gta aag atc agt ggagga cct cac att tcc tac cca ccc 3373 His His Met Val Lys Ile Ser Gly GlyPro His Ile Ser Tyr Pro Pro 290 295 300 ctg cat gag tgg gtt ttg aga gagggg gaa gag tga gtctgagcac 3419 Leu His Glu Trp Val Leu Arg Glu Gly GluGlu 305 310 gagttgcagc cagggccagt gggagggggt ctgggccagt gcaccttccggggccgcatc 3479 ccttagtttc cactgcctcc tgtgacgtga ggcccattct tcactctttgaagcgagcag 3539 tcagcattct tagtagtggg tttctgttct gttggatgac tttgagattattctttgttt 3599 cctgttggag ttgttcaaat gttcctttta acggatggtt gaatgagcgtcagcatccag 3659 gtttatgaat gacagtagtc acacatagtg ctgtttatat agtttaggagtaagagtctt 3719 gttttttact caaattggga aatccattcc attttgtgaa ttgtgacataataatagcag 3779 tggtaaaagt atttgcttaa aattgtgagc gaattagcaa taacatacatgagataactc 3839 aagaaatcaa aagatagttg attcttgcct tgtacctcaa tctattctgtaaaattaaac 3899 aaatatgcaa accaggattt ccttgacttc tttgagaatg caagcgaaattaaatctgaa 3959 taaataattc ttcctcttca ctggctcgtt tcttttccgt tcactcagcatctgctctgt 4019 gggaggccct gggttagtag tggggatgct aaggtaagcc agactcacgcctacccatag 4079 ggctgtagag cctaggacct gcagtcatat aattaaggtg gtgagaagtcctgtaagatg 4139 tagaggaaat gtaagagagg ggtgagggtg tggcgctccg ggtgagagtagtggagtgtc 4199 agtgc 4204 2 314 PRT Homo sapiens 2 Met Pro Leu Glu GlnArg Ser Gln His Cys Lys Pro Glu Glu Gly Leu 1 5 10 15 Glu Ala Arg GlyGlu Ala Leu Gly Leu Val Gly Ala Gln Ala Pro Ala 20 25 30 Thr Glu Glu GlnGlu Ala Ala Ser Ser Ser Ser Thr Leu Val Glu Val 35 40 45 Thr Leu Gly GluVal Pro Ala Ala Glu Ser Pro Asp Pro Pro Gln Ser 50 55 60 Pro Gln Gly AlaSer Ser Leu Pro Thr Thr Met Asn Tyr Pro Leu Trp 65 70 75 80 Ser Gln SerTyr Glu Asp Ser Ser Asn Gln Glu Glu Glu Gly Pro Ser 85 90 95 Thr Phe ProAsp Leu Glu Ser Glu Phe Gln Ala Ala Leu Ser Arg Lys 100 105 110 Val AlaGlu Leu Val His Phe Leu Leu Leu Lys Tyr Arg Ala Arg Glu 115 120 125 ProVal Thr Lys Ala Glu Met Leu Gly Ser Val Val Gly Asn Trp Gln 130 135 140Tyr Phe Phe Pro Val Ile Phe Ser Lys Ala Ser Ser Ser Leu Gln Leu 145 150155 160 Val Phe Gly Ile Glu Leu Met Glu Val Asp Pro Ile Gly His Leu Tyr165 170 175 Ile Phe Ala Thr Cys Leu Gly Leu Ser Tyr Asp Gly Leu Leu GlyAsp 180 185 190 Asn Gln Ile Met Pro Lys Ala Gly Leu Leu Ile Ile Val LeuAla Ile 195 200 205 Ile Ala Arg Glu Gly Asp Cys Ala Pro Glu Glu Lys IleTrp Glu Glu 210 215 220 Leu Ser Val Leu Glu Val Phe Glu Gly Arg Glu AspSer Ile Leu Gly 225 230 235 240 Asp Pro Lys Lys Leu Leu Thr Gln His PheVal Gln Glu Asn Tyr Leu 245 250 255 Glu Tyr Arg Gln Val Pro Gly Ser AspPro Ala Cys Tyr Glu Phe Leu 260 265 270 Trp Gly Pro Arg Ala Leu Val GluThr Ser Tyr Val Lys Val Leu His 275 280 285 His Met Val Lys Ile Ser GlyGly Pro His Ile Ser Tyr Pro Pro Leu 290 295 300 His Glu Trp Val Leu ArgGlu Gly Glu Glu 305 310 3 16 PRT Homo sapiens 3 Gly Ser Asp Pro Ala CysTyr Glu Phe Leu Trp Gly Pro Arg Ala Leu 1 5 10 15 4 16 PRT Homo sapiens4 Ala Cys Tyr Glu Phe Leu Trp Gly Pro Arg Ala Leu Val Glu Thr Ser 1 5 1015 5 48 DNA Homo sapiens 5 ggcagtgatc ctgcatgtta tgaattcctg tggggtccaagggccctc 48 6 48 DNA Homo sapiens 6 gcatgttatg aattcctgtg gggtccaagggccctcgttg aaaccagc 48 7 14 PRT Homo sapiens 7 Ala Cys Tyr Glu Phe LeuTrp Gly Pro Arg Ala Leu Val Glu 1 5 10 8 13 PRT Homo sapiens 8 Ala CysTyr Glu Phe Leu Trp Gly Pro Arg Ala Leu Val 1 5 10 9 12 PRT Homo sapiens9 Ala Cys Tyr Glu Phe Leu Trp Gly Pro Arg Ala Leu 1 5 10 10 11 PRT Homosapiens 10 Ala Cys Tyr Glu Phe Leu Trp Gly Pro Arg Ala 1 5 10 11 10 PRTHomo sapiens 11 Ala Cys Tyr Glu Phe Leu Trp Gly Pro Arg 1 5 10 12 13 PRTHomo sapiens 12 Cys Tyr Glu Phe Leu Trp Gly Pro Arg Ala Leu Val Glu 1 510 13 12 PRT Homo sapiens 13 Tyr Glu Phe Leu Trp Gly Pro Arg Ala Leu ValGlu 1 5 10 14 11 PRT Homo sapiens 14 Glu Phe Leu Trp Gly Pro Arg Ala LeuVal Glu 1 5 10 15 42 DNA Homo sapiens 15 gcatgttatg aattcctgtggggtccaagg gccctcgttg aa 42 16 15 PRT Homo sapiens 16 Ala Cys Tyr GluPhe Leu Trp Gly Pro Arg Ala Leu Val Glu Thr 1 5 10 15 17 24 DNA Homosapiens 17 ccctcatgag ggcaggtgcc accg 24 18 31 DNA Homo sapiens 18cccagatctc tagaggattc ccctgttcca c 31

We claim:
 1. An isolated MAGE-A3 HLA class II-binding peptide comprisingan amino acid sequence selected from the group consisting of SEQ IDNO:3, SEQ ID NO:4 and SEQ ID NO:7, or a functional variant thereofcomprising 1-10 amino acid additions, substitutions or deletions,wherein the HLA class II binding peptide does not include the fulllength MAGE-A3 protein comprising the amino acid sequence of SEQ IDNO:2.
 2. The isolated HLA class II-binding peptide of claim 1 whereinthe isolated peptide consists essentially of an amino acid sequenceselected from the group consisting of SEQ ID NO:3, SEQ ID NO:4 and SEQID NO:7.
 3. The isolated HLA class II-binding peptide of claim 1 whereinthe isolated peptide consists of an amino acid sequence selected fromthe group consisting of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO: 7 and SEQID NO:
 16. 4. The isolated HLA class II-binding peptide of claim 3wherein the isolated peptide consists of the amino acid sequence setforth as SEQ ID NO:4.
 5. The isolated HLA class II-binding peptide ofclaim 1, wherein the isolated peptide comprises an endosomal targetingsignal.
 6. The isolated HLA class II-binding peptide of claim 5, whereinthe endosomal targeting signal comprises an endosomal targeting portionof human invariant chain Ii.
 7. The isolated HLA class II-bindingpeptide of claim 1 wherein the isolated peptide is non-hydrolyzable. 8.The isolated HLA class II-binding peptide of claim 7 wherein theisolated peptide is selected from the group consisting of peptidescomprising D-amino acids, peptides comprising a -psi[CH₂NH]-reducedamide peptide bond, peptides comprising a -psi[COCH₂]-ketomethylenepeptide bond, peptides comprising a -psi[CH(CN)NH]-(cyanomethylene)aminopeptide bond, peptides comprising a -psi[CH₂CH(OH)]-hydroxyethylenepeptide bond, peptides comprising a -psi[CH₂O]-peptide bond, andpeptides comprising a -psi[CH₂S]-thiomethylene peptide bond.
 9. Acomposition comprising an isolated HLA class I-binding peptide and anisolated MAGE-A3 HLA class II-binding peptide, wherein the isolatedMAGE-A3 HLA class II-binding peptide comprises an amino acid sequenceselected from the group consisting of SEQ ID NO:3, SEQ ID NO:4 and SEQID NO:7, or a functional variant thereof, and wherein the HLA class IIbinding peptide does not include the full length MAGE-A3 protein. 10.The composition of claim 9, wherein the HLA class I-binding peptide andthe MAGE-A3 HLA class II-binding peptide are combined as a polytopepolypeptide.
 11. The composition of claim 9, wherein the isolatedMAGE-A3 HLA class II-binding peptide consists essentially of an aminoacid sequence selected from the group consisting of SEQ ID NO:3, SEQ IDNO:4 and SEQ ID NO:7.
 12. The composition of claim 9, wherein theisolated MAGE-A3 HLA class II-binding peptide consists of an amino acidsequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:4,SEQ ID NO:7 and SEQ ID NO:
 16. 13. The composition of claim 12, whereinthe isolated MAGE-A3 HLA class II-binding peptide consists of the aminoacid sequence set forth as SEQ ID NO:4.
 14. The composition of claim 9,wherein the isolated MAGE-A3 HLA class II-binding peptide comprises anendosomal targeting signal.
 15. The composition of claim 14, wherein theendosomal targeting signal comprises an endosomal targeting portion ofhuman invariant chain Ii.
 16. A composition comprising one or more ofthe isolated MAGE-A3 HLA class II-binding peptides of claim Al complexedwith one or more isolated HLA class II molecules.
 17. The composition ofclaim 16, wherein the number of isolated MAGE-A3 HLA class II-bindingpeptides and the number of isolated HLA class II molecules are equal.18. The method of claim 17, wherein the isolated MAGE-A3 HLA classII-binding peptides and the isolated MAGE-A3 HLA class II molecules arecoupled as a tetrameric molecule of individual isolated MAGE-A3 HLAclass II-binding peptides bound to individual isolated HLA class IImolecules.
 19. The method of claim 16, wherein the HLA class IImolecules are DR1 molecules.
 20. An isolated nucleic acid encoding apeptide selected from the group consisting of the peptide of claim 1,the peptide of claim 2, the peptide of claim 3 and the peptide of claim5, wherein the nucleic acid molecule does not encode full length MAGE-A3protein.
 21. The isolated nucleic acid of claim 20, wherein the nucleicacid comprises a nucleotide sequence selected from the group consistingof SEQ ID NO:5, SEQ ID NO:6 and SEQ ID NO:15.
 22. An expression vectorcomprising the isolated nucleic acid of claim 20 operably linked to apromoter.
 23. The expression vector of claim 22 wherein the nucleic acidcomprises a nucleotide sequence selected from the group consisting ofSEQ ID NO:5, SEQ ID NO:6 and SEQ ID NO:15.
 24. The expression vector ofclaim 22 or 23 further comprising a nucleic acid which encodes anHLA-DR1 molecule.
 25. A host cell transfected or transformed with anexpression vector selected from the group consisting of the expressionvector of claim 22 and the expression vector of claim
 23. 26. A hostcell transfected or transformed with the expression vector of claim 24.27. A host cell transfected or transformed with an expression vectorselected from the group of the expression vector of claim 22 and theexpression vector of claim 23, and wherein the host cell expresses anHLA-DR1 molecule.
 28. A method for enriching selectively a population ofT lymphocytes with CD4⁺ T lymphocytes specific for a MAGE-A3 HLA classII-binding peptide comprising: contacting an isolated population of Tlymphocytes with an agent presenting a complex of the MAGE-A3 HLA classII-binding peptide and an HLA class II molecule in an amount sufficientto selectively enrich the isolated population of T lymphocytes with theCD4⁺ T lymphocytes, wherein the MAGE-A3 HLA class II-binding peptidecomprises an amino acid sequence selected from the group consisting ofSEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:7, or a functional variantthereof.
 29. The method of claim 28 wherein the MAGE-A3 HLA classII-binding peptide consists essentially of an amino acid sequenceselected from the group consisting of SEQ ID NO:3, SEQ ID NO:4 and SEQID NO:7.
 30. The method of claim 28 wherein the MAGE-A3 HLA classII-binding peptide consists of an amino acid sequence selected from thegroup consisting of SEQ ID NO:3, SEQ ID NO: 4, SEQ ID NO:7 and SEQ IDNO:16.
 31. The method of claim 30 wherein the MAGE-A3 HLA classII-binding peptide consists of the amino acid sequence set forth as SEQID NO:4.
 32. The method of claim 28 wherein the HLA class II molecule isan HLA-DR1 molecule.
 33. The method of claim 28, wherein the MAGE-A3 HLAclass II binding peptide comprises an endosomal targeting portion ofhuman invariant chain Ii.
 34. A method for diagnosing a disordercharacterized by expression of MAGE-A3 comprising: contacting abiological sample isolated from a subject with an agent that is specificfor the MAGE-A3 HLA class II binding peptide, and determining theinteraction between the agent and the MAGE-A3 HLA class II bindingpeptide as a determination of the disorder, wherein the MAGE-A3 HLAclass II-binding peptide comprises an amino acid sequence selected fromthe group consisting of SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:7, or afunctional variant thereof.
 35. The method of claim 34, wherein theMAGE-A3 HLA class II-binding peptide consists essentially of an aminoacid sequence selected from the group consisting of SEQ ID NO:3, SEQ IDNO:4 and SEQ ID NO:7.
 36. The method of claim 34, wherein the MAGE-A3HLA class II-binding peptide consists of an amino acid sequence selectedfrom the group consisting of SEQ ID NO:3, SEQ ID NO: 4, SEQ ID NO:7 andSEQ ID NO:16.
 37. The method of claim 36, wherein the MAGE-A3 HLA classII-binding peptide consists of the amino acid sequence set forth as SEQID NO:4.
 38. The method of claim 34, wherein the agent is an antibody oran antigen binding fragment thereof.
 39. A method for diagnosing adisorder characterized by expression of a MAGE-A3 HLA class II-bindingpeptide which forms a complex with an HLA class II molecule, comprising:contacting a biological sample isolated from a subject with an agentthat binds the complex; and determining binding between the complex andthe agent as a determination of the disorder, wherein the HLA class IImolecule is an HLA-DR1 and the MAGE-A3 HLA class II-binding peptidecomprises an amino acid sequence selected from the group consisting ofSEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:7, or a functional variantthereof.
 40. The method of claim 39, wherein the MAGE-A3 HLA classII-binding peptide consists essentially of an amino acid sequenceselected from the group consisting of SEQ ID NO:3, SEQ ID NO:4 and SEQID NO:7.
 41. The method of claim 39, wherein the MAGE-A3 HLA classII-binding peptide consists of an amino acid sequence selected from thegroup consisting of SEQ ID NO:3, SEQ ID NO: 4, SEQ ID NO:7 and SEQ IDNO:
 16. 42. The method of claim 41, wherein the MAGE-A3 HLA classII-binding peptide consists of the amino acid sequence set forth as SEQID NO:4.
 43. A method for treating a subject having a disordercharacterized by expression of MAGE-A3, comprising: administering to thesubject an amount of a MAGE-A3 HLA class II-binding peptide effective toameliorate the disorder, wherein the MAGE-A3 HLA class II-bindingpeptide comprises an amino acid sequence selected from the groupconsisting of SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:7, or a functionalvariant thereof.
 44. The method of claim 43, wherein the MAGE-A3 HLAclass II binding peptide comprises an endosomal targeting signal. 45.The method of claim 44, wherein the endosomal targeting signal comprisesan endosomal targeting portion of human invariant chain Ii.
 46. Themethod of claim 43, wherein the MAGE-A3 HLA class II-binding peptideconsists essentially of an amino acid sequence selected from the groupconsisting of SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:7.
 47. The methodof claim 43, wherein the MAGE-A3 HLA class II-binding peptide consistsof an amino acid sequence selected from the group consisting of SEQ IDNO:3, SEQ ID NO: 4, SEQ ID NO:7 and SEQ ID NO:16.
 48. The method ofclaim 47, wherein the MAGE-A3 HLA class II-binding peptide consists ofthe amino acid sequence set forth as SEQ ID NO:4.
 49. A method fortreating a subject having a disorder characterized by expression ofMAGE-A3, comprising: administering to the subject an amount of a HLAclass I-binding peptide and an amount of a MAGE-A3 HLA class II-bindingpeptide effective to ameliorate the disorder, wherein the MAGE-A3 HLAclass II-binding peptide comprises an amino acid sequence selected fromthe group consisting of SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:7, or afunctional variant thereof.
 50. The method of claim 49, wherein the HLAclass I-binding peptide and the MAGE-A3 HLA class II-binding peptide arecombined as a polytope polypeptide.
 51. The method of claim 49, whereinthe HLA class I-binding peptide is a MAGE-A3 HLA class I-bindingpeptide.
 52. The method of claim 49, wherein the MAGE-A3 HLA class IIbinding peptide comprises an endosomal targeting signal.
 53. The methodof claim 52, wherein the endosomal targeting signal comprises anendosomal targeting portion of human invariant chain Ii.
 54. The methodof claim 49, wherein the MAGE-A3 HLA class II-binding peptide consistsessentially of an amino acid sequence selected from the group consistingof SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:7.
 55. The method of claim 49,wherein MAGE-A3 HLA class II-binding peptide consists of an amino acidsequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:4,SEQ ID NO:7 and SEQ ID NO:16.
 56. The method of claim 55, wherein theMAGE-A3 HLA class II-binding peptide consists of the amino acid sequenceset forth as SEQ ID NO:4.
 57. A method for treating a subject having adisorder characterized by expression of MAGE-A3, comprising:administering to the subject an amount of an agent which enrichesselectively in the subject the presence of complexes of an HLA class IImolecule and a MAGE-A3 HLA class II-binding peptide, sufficient toameliorate the disorder, wherein the HLA class II molecule is an HLA-DR1molecule and the MAGE-A3 HLA class II-binding peptide comprises an aminoacid sequence selected from the group consisting of SEQ ID NO:3, SEQ IDNO:4 and SEQ ID NO:7, or a functional variant thereof.
 58. The method ofclaim 57, wherein the MAGE-A3 HLA class II-binding peptide consistsessentially of an amino acid sequence selected from the group consistingof SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:7.
 59. The method of claim 57,wherein the MAGE-A3 HLA class II-binding peptide consists of an aminoacid sequence selected from the group consisting of SEQ ID NO:3, SEQ IDNO: 4, SEQ ID NO:7 and SEQ ID NO:16.
 60. The method of claim 59, whereinthe MAGE-A3 HLA class II-binding peptide consists of the amino acidsequence set forth as SEQ ID NO:4.
 61. The method of claim 57, whereinthe agent comprises a MAGE-A3 HLA class II binding peptide.
 62. Themethod of claim 61, wherein the MAGE-A3 HLA class II binding peptidecomprises an endosomal targeting signal.
 63. The method of claim 62,wherein the endosomal targeting signal comprises an endosomal targetingportion of human invariant chain Ii.
 64. A method for treating a subjecthaving a disorder characterized by expression of MAGE-A3, comprising:administering to the subject an amount of autologous CD4⁺ T lymphocytessufficient to ameliorate the disorder, wherein the CD4⁺ T lymphocytesare specific for complexes of an HLA class II molecule and a MAGE-A3 HLAclass II-binding peptide, wherein the HLA class II molecule is anHLA-DR1 molecule and the MAGE-A3 HLA class II-binding peptide comprisesan amino acid sequence selected from the group consisting of SEQ IDNO:3, SEQ ID NO:4 and SEQ ID NO:7, or a functional variant thereof. 65.The method of claim 64, wherein the MAGE-A3 HLA class II-binding peptideconsists essentially of an amino acid sequence selected from the groupconsisting of SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:7.
 66. The methodof claim 64, wherein the MAGE-A3 HLA class II-binding peptide consistsof an amino acid sequence selected from the group consisting of SEQ IDNO:3, SEQ ID NO: 4, SEQ ID NO:7 and SEQ ID NO:16.
 67. The method ofclaim 66, wherein the MAGE-A3 HLA class II-binding peptide consists ofthe amino acid sequence set forth as SEQ ID NO:4.
 68. A method foridentifying functional variants of a MAGE-A3 HLA class II bindingpeptide, comprising selecting a MAGE-A3 HLA class II binding peptidewhich comprisies an amino acid sequence selected from the groupconsisting of SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:7, an HLA class IIbinding molecule which binds the MAGE-A3 HLA class II binding peptide,and a T cell which is stimulated by the MAGE-A3 HLA class II bindingpeptide presented by the HLA class II binding molecule; mutating a firstamino acid residue of the MAGE-A3 HLA class II binding peptide toprepare a variant peptide; and determining the binding of the variantpeptide to HLA class II binding molecule and the stimulation of the Tcell, wherein binding of the variant peptide to the HLA class II bindingmolecule and stimulation of the T cell by the variant peptide presentedby the HLA class II binding molecule indicates that the variant peptideis a functional variant.
 69. The method of claim 68, further comprisingthe step of comparing the stimulation of the T cell by the MAGE-A3 HLAclass II binding peptide and the stimulation of the T cell by thefunctional variant as a determination of the effectiveness of thestimulation of the T cell by the functional variant.
 70. An isolatedpolypeptide which binds selectively a polypeptide of claim 2, providedthat the isolated polypeptide is not an HLA class II molecule.
 71. Theisolated polypeptide of claim 70, wherein the isolated polypeptide is anantibody.
 72. The antibody of claim 71, wherein the antibody is amonoclonal antibody.
 73. The antibody of claim 72, wherein themonoclonal antibody is a human antibody, a humanized antibody, achimeric antibody or a single chain antibody.
 74. The isolatedpolypeptide of claim 70, wherein the isolated polypeptide is an antibodyfragment selected from the group consisting of a Fab fragment, a F(ab)₂fragment, a Fv fragment or a fragment including a CDR3 region selectivefor a MAGE-A3 HLA class II-binding peptide.
 75. An isolated CD4⁺ Tlymphocyte which selectively binds a complex of an HLA class II moleculeand a MAGE-A3 HLA class II-binding peptide, wherein the HLA class IImolecule is an HLA-DR1 molecule and wherein the MAGE-A3 HLA classII-binding peptide comprises an amino acid sequence selected from thegroup consisting of SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:7, or afunctional variant thereof.
 76. The isolated CD4⁺ T lymphocyte of claim75 wherein the MAGE-A3 HLA class II-binding peptide consists essentiallyof an amino acid sequence selected from the group consisting of SEQ IDNO:3, SEQ ID NO:4 and SEQ ID NO:7.
 77. The isolated CD4⁺ T lymphocyte ofclaim 75 wherein the MAGE-A3 HLA class II-binding peptide consists of anamino acid sequence selected from the group consisting of SEQ ID NO:3,SEQ ID NO: 4, SEQ ID NO:7 and SEQ ID NO:16.
 78. The isolated CD4⁺ Tlymphocyte of claim 77 wherein the MAGE-A3 HLA class II-binding peptideconsists of the amino acid sequence set forth as SEQ ID NO:4.
 79. Anisolated antigen presenting cell which comprises a complex of an HLAclass 11 molecule and a MAGE-A3 HLA class II-binding peptide, whereinthe HLA class II molecule is an HLA-DR1 molecule and wherein the MAGE-A3HLA class II-binding peptide comprises an amino acid sequence selectedfrom the group consisting of SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:7.80. The isolated antigen presenting cell of claim 79 wherein the MAGE-A3HLA class II-binding peptide consists essentially of an amino acidsequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:4and SEQ ID NO:7.
 81. The isolated antigen presenting cell of claim 79wherein the MAGE-A3 HLA class II-binding peptide consists of an aminoacid sequence selected from the group consisting of SEQ ID NO:3, SEQ IDNO: 4, SEQ ID NO:7 and SEQ ID NO:16.
 82. The isolated antigen presentingcell of claim 81 wherein the MAGE-A3 HLA class II-binding peptideconsists of the amino acid sequence set forth as SEQ ID NO:4.